<?xml version="1.0" encoding="utf-8"?>
<!--XBRL document created with Toppan Merrill Bridge  11.2.0.5 -->
<!--Based on XBRL 2.1-->
<!--Created on: 3/16/2026 10:36:50 PM-->
<!--Modified on: 3/16/2026 10:36:50 PM-->
<link:linkbase xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd" xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xbrli="http://www.xbrl.org/2003/instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt">
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DocumentDocumentAndEntityInformation" roleURI="http://www.bicycletherapeutics.com/role/DocumentDocumentAndEntityInformation" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#StatementConsolidatedStatementsOfShareholdersEquity" roleURI="http://www.bicycletherapeutics.com/role/StatementConsolidatedStatementsOfShareholdersEquity" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsTables" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsTables" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureNatureOfBusinessAndBasisOfPresentationShareActivityDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureNatureOfBusinessAndBasisOfPresentationShareActivityDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSummaryOfSignificantAccountingPoliciesPropertyAndEquipmentGeographicDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSummaryOfSignificantAccountingPoliciesPropertyAndEquipmentGeographicDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSummaryOfSignificantAccountingPoliciesResearchAndDevelopmentIncentivesAndReceivableDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSummaryOfSignificantAccountingPoliciesResearchAndDevelopmentIncentivesAndReceivableDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureFairValueOfFinancialAssetsAndLiabilitiesFinancialAssetsAndLiabilitiesMeasuredAtFairValueDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureFairValueOfFinancialAssetsAndLiabilitiesFinancialAssetsAndLiabilitiesMeasuredAtFairValueDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosurePropertyAndEquipmentNetDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosurePropertyAndEquipmentNetDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureAccruedExpensesAndOtherCurrentLiabilitiesAdditionalInformationDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureAccruedExpensesAndOtherCurrentLiabilitiesAdditionalInformationDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureDebtDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureDebtDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureOrdinarySharesDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureOrdinarySharesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureShareBasedCompensationDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureShareBasedCompensationShareBasedCompensationExpenseDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationShareBasedCompensationExpenseDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureShareBasedCompensationShareOptionsDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationShareOptionsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureShareBasedCompensationAssumptionsUsedInBlackScholesOptionPricingModelToDetermineFairValueOfShareOptionsDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationAssumptionsUsedInBlackScholesOptionPricingModelToDetermineFairValueOfShareOptionsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureShareBasedCompensationRestrictedShareUnitsDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationRestrictedShareUnitsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsCollaborationRevenuesDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsCollaborationRevenuesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsBayerCollaborationAgreementDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsBayerCollaborationAgreementDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsBayerCollaborationAgreementAccountingAnalysisDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsBayerCollaborationAgreementAccountingAnalysisDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsNovartisCollaborationAgreementDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsNovartisCollaborationAgreementDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsIonisEvaluationAndOptionAgreementDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsIonisEvaluationAndOptionAgreementDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsIonisCollaborationAgreementDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsIonisCollaborationAgreementDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsIonisSharePurchaseAgreementDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsIonisSharePurchaseAgreementDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsIonisAccountingAnalysisDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsIonisAccountingAnalysisDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsGenentechCollaborationAgreementDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsGenentechCollaborationAgreementDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsSummaryOfContractAssetsAndLiabilitiesDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsSummaryOfContractAssetsAndLiabilitiesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSignificantAgreementsCancerResearchUkDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsCancerResearchUkDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureIncomeTaxesReconciliationOfBenefitForIncomeTaxesDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureIncomeTaxesReconciliationOfBenefitForIncomeTaxesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureIncomeTaxesDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureIncomeTaxesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureCommitmentsAndContingenciesOperatingLeasesAndFinanceLeasesDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureCommitmentsAndContingenciesOperatingLeasesAndFinanceLeasesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureCommitmentsAndContingenciesLeasesMaturitiesOfOperatingLeasesDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureCommitmentsAndContingenciesLeasesMaturitiesOfOperatingLeasesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureNetLossPerShareSecuritiesExcludedFromDilutedPerShareCalculationDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureNetLossPerShareSecuritiesExcludedFromDilutedPerShareCalculationDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureBenefitPlansDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureBenefitPlansDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureRelatedPartyTransactionsDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureRelatedPartyTransactionsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSegmentsAndGeographicInformationDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSegmentsAndGeographicInformationDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSegmentsAndGeographicInformationSignificantSegmentExpensesDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSegmentsAndGeographicInformationSignificantSegmentExpensesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="bcyc-20251231.xsd#DisclosureSubsequentEventsDetails" roleURI="http://www.bicycletherapeutics.com/role/DisclosureSubsequentEventsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/IndividualsOnly" xlink:href="https://xbrl.sec.gov/ecd/2025/ecd-2025.xsd#IndividualsOnly" xlink:type="simple" />
  <link:arcroleRef arcroleURI="http://www.esma.europa.eu/xbrl/esef/arcrole/wider-narrower" xlink:type="simple" xlink:href="http://www.xbrl.org/lrr/arcrole/esma-arcrole-2018-11-21.xsd#wider-narrower" />
  <link:roleRef roleURI="http://www.bicycletherapeutics.com/role/DisclosureStandardAndCustomAxisDomainDefaults" xlink:href="bcyc-20251231.xsd#DisclosureStandardAndCustomAxisDomainDefaults" xlink:type="simple" />
  <link:roleRef roleURI="http://fasb.org/us-gaap/role/eedm/ExtensibleEnumerationLists" xlink:type="simple" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-roles-2025.xsd#eedm" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" />
  <link:definitionLink xlink:type="extended" xlink:role="http://fasb.org/us-gaap/role/eedm/ExtensibleEnumerationLists" xlink:title="Extensible Enumeration Domain Members">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LiabilitiesCurrentAbstract" xlink:label="us-gaap_LiabilitiesCurrentAbstract_1" xlink:title="us-gaap_LiabilitiesCurrentAbstract_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:label="us-gaap_AccruedLiabilitiesCurrent_1" xlink:title="us-gaap_AccruedLiabilitiesCurrent_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1" xlink:to="us-gaap_AccruedLiabilitiesCurrent_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:us-gaap_LiabilitiesCurrentAbstract_1 To us-gaap_AccruedLiabilitiesCurrent_1" order="1" priority="0" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetsAbstract" xlink:label="us-gaap_AssetsAbstract_1" xlink:title="us-gaap_AssetsAbstract_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="us-gaap_PropertyPlantAndEquipmentNet_1" xlink:title="us-gaap_PropertyPlantAndEquipmentNet_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_AssetsAbstract_1" xlink:to="us-gaap_PropertyPlantAndEquipmentNet_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:us-gaap_AssetsAbstract_1 To us-gaap_PropertyPlantAndEquipmentNet_1" order="1" priority="0" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LiabilitiesAbstract" xlink:label="us-gaap_LiabilitiesAbstract" xlink:title="us-gaap_LiabilitiesAbstract" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:label="us-gaap_AccruedLiabilitiesCurrent_2" xlink:title="us-gaap_AccruedLiabilitiesCurrent_2" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_LiabilitiesAbstract" xlink:to="us-gaap_AccruedLiabilitiesCurrent_2" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:us-gaap_LiabilitiesAbstract To us-gaap_AccruedLiabilitiesCurrent_2" order="1" priority="0" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherLiabilitiesNoncurrent" xlink:label="us-gaap_OtherLiabilitiesNoncurrent_1" xlink:title="us-gaap_OtherLiabilitiesNoncurrent_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_LiabilitiesAbstract" xlink:to="us-gaap_OtherLiabilitiesNoncurrent_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:us-gaap_LiabilitiesAbstract To us-gaap_OtherLiabilitiesNoncurrent_1" order="2" priority="0" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementAbstract" xlink:label="us-gaap_IncomeStatementAbstract" xlink:title="us-gaap_IncomeStatementAbstract" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:title="us-gaap_ResearchAndDevelopmentExpense" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_IncomeStatementAbstract" xlink:to="us-gaap_ResearchAndDevelopmentExpense" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:us-gaap_IncomeStatementAbstract To us-gaap_ResearchAndDevelopmentExpense" order="1" priority="0" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherAssets" xlink:label="us-gaap_OtherAssets" xlink:title="us-gaap_OtherAssets" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedCashAndCashEquivalents" xlink:label="us-gaap_RestrictedCashAndCashEquivalents_2" xlink:title="us-gaap_RestrictedCashAndCashEquivalents_2" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_LiabilitiesAbstract" xlink:to="us-gaap_RestrictedCashAndCashEquivalents_2" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:us-gaap_LiabilitiesAbstract To us-gaap_RestrictedCashAndCashEquivalents_2" order="3" priority="0" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedCashAndCashEquivalents" xlink:label="us-gaap_RestrictedCashAndCashEquivalents_3" xlink:title="us-gaap_RestrictedCashAndCashEquivalents_3" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_AssetsAbstract_1" xlink:to="us-gaap_RestrictedCashAndCashEquivalents_3" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:us-gaap_AssetsAbstract_1 To us-gaap_RestrictedCashAndCashEquivalents_3" order="2" priority="0" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:label="us-gaap_PrepaidExpenseAndOtherAssetsCurrent_1" xlink:title="us-gaap_PrepaidExpenseAndOtherAssetsCurrent_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_AssetsAbstract_1" xlink:to="us-gaap_PrepaidExpenseAndOtherAssetsCurrent_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:us-gaap_AssetsAbstract_1 To us-gaap_PrepaidExpenseAndOtherAssetsCurrent_1" order="3" priority="0" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherAssetsNoncurrent" xlink:label="us-gaap_OtherAssetsNoncurrent_1" xlink:title="us-gaap_OtherAssetsNoncurrent_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_AssetsAbstract_1" xlink:to="us-gaap_OtherAssetsNoncurrent_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:us-gaap_AssetsAbstract_1 To us-gaap_OtherAssetsNoncurrent_1" order="14" priority="0" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureStandardAndCustomAxisDomainDefaults" xlink:type="extended" xlink:title="99900 - Disclosure - Standard And Custom Axis Domain Defaults">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_1" xlink:title="us-gaap_StatementClassOfStockAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1" xlink:title="us-gaap_ClassOfStockDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_StatementClassOfStockAxis_1" xlink:to="us-gaap_ClassOfStockDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_StatementClassOfStockAxis_1 To us-gaap_ClassOfStockDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis_1" xlink:title="us-gaap_StatementEquityComponentsAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain_1" xlink:title="us-gaap_EquityComponentDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_StatementEquityComponentsAxis_1" xlink:to="us-gaap_EquityComponentDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_StatementEquityComponentsAxis_1 To us-gaap_EquityComponentDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis_1" xlink:title="srt_TitleOfIndividualAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain_1" xlink:title="srt_TitleOfIndividualWithRelationshipToEntityDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_TitleOfIndividualAxis_1" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_TitleOfIndividualAxis_1 To srt_TitleOfIndividualWithRelationshipToEntityDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis_1" xlink:title="us-gaap_TypeOfArrangementAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1" xlink:title="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_TypeOfArrangementAxis_1" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_TypeOfArrangementAxis_1 To us-gaap_ArrangementsAndNonarrangementTransactionsMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis_1" xlink:title="us-gaap_SubsidiarySaleOfStockAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain_1" xlink:title="us-gaap_SaleOfStockNameOfTransactionDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_SubsidiarySaleOfStockAxis_1" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_SubsidiarySaleOfStockAxis_1 To us-gaap_SaleOfStockNameOfTransactionDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis" xlink:label="us-gaap_LongtermDebtTypeAxis_1" xlink:title="us-gaap_LongtermDebtTypeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain_1" xlink:title="us-gaap_LongtermDebtTypeDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_LongtermDebtTypeAxis_1" xlink:to="us-gaap_LongtermDebtTypeDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_LongtermDebtTypeAxis_1 To us-gaap_LongtermDebtTypeDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis_1" xlink:title="us-gaap_FairValueByFairValueHierarchyLevelAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1" xlink:title="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis_1" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_FairValueByFairValueHierarchyLevelAxis_1 To us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByLiabilityClassAxis" xlink:label="us-gaap_FairValueByLiabilityClassAxis_1" xlink:title="us-gaap_FairValueByLiabilityClassAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" xlink:label="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_1" xlink:title="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_FairValueByLiabilityClassAxis_1" xlink:to="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_FairValueByLiabilityClassAxis_1 To us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByMeasurementFrequencyAxis" xlink:label="us-gaap_FairValueByMeasurementFrequencyAxis_1" xlink:title="us-gaap_FairValueByMeasurementFrequencyAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain_1" xlink:title="us-gaap_FairValueMeasurementFrequencyDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis_1" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_FairValueByMeasurementFrequencyAxis_1 To us-gaap_FairValueMeasurementFrequencyDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis_1" xlink:title="us-gaap_PropertyPlantAndEquipmentByTypeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain_1" xlink:title="us-gaap_PropertyPlantAndEquipmentTypeDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis_1" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_PropertyPlantAndEquipmentByTypeAxis_1 To us-gaap_PropertyPlantAndEquipmentTypeDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VariableRateAxis" xlink:label="us-gaap_VariableRateAxis_1" xlink:title="us-gaap_VariableRateAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain_1" xlink:title="us-gaap_VariableRateDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_VariableRateAxis_1" xlink:to="us-gaap_VariableRateDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_VariableRateAxis_1 To us-gaap_VariableRateDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementScenarioAxis" xlink:label="srt_StatementScenarioAxis_1" xlink:title="srt_StatementScenarioAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ScenarioUnspecifiedDomain" xlink:label="srt_ScenarioUnspecifiedDomain_1" xlink:title="srt_ScenarioUnspecifiedDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_StatementScenarioAxis_1" xlink:to="srt_ScenarioUnspecifiedDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_StatementScenarioAxis_1 To srt_ScenarioUnspecifiedDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis_1" xlink:title="us-gaap_PlanNameAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain_1" xlink:title="us-gaap_PlanNameDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_PlanNameAxis_1" xlink:to="us-gaap_PlanNameDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_PlanNameAxis_1 To us-gaap_PlanNameDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis_1" xlink:title="srt_RangeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember_1" xlink:title="srt_RangeMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_RangeAxis_1" xlink:to="srt_RangeMember_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_RangeAxis_1 To srt_RangeMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis_1" xlink:title="us-gaap_AwardTypeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_1" xlink:title="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_AwardTypeAxis_1" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_AwardTypeAxis_1 To us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GranteeStatusAxis" xlink:label="us-gaap_GranteeStatusAxis_1" xlink:title="us-gaap_GranteeStatusAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain_1" xlink:title="us-gaap_GranteeStatusDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_GranteeStatusAxis_1" xlink:to="us-gaap_GranteeStatusDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_GranteeStatusAxis_1 To us-gaap_GranteeStatusDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis_1" xlink:title="us-gaap_SubsequentEventTypeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain_1" xlink:title="us-gaap_SubsequentEventTypeDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_SubsequentEventTypeAxis_1" xlink:to="us-gaap_SubsequentEventTypeDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_SubsequentEventTypeAxis_1 To us-gaap_SubsequentEventTypeDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis" xlink:label="srt_StatementGeographicalAxis_1" xlink:title="srt_StatementGeographicalAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain_1" xlink:title="srt_SegmentGeographicalDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_StatementGeographicalAxis_1" xlink:to="srt_SegmentGeographicalDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_StatementGeographicalAxis_1 To srt_SegmentGeographicalDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingAxis" xlink:label="us-gaap_VestingAxis_1" xlink:title="us-gaap_VestingAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain_1" xlink:title="us-gaap_VestingDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_VestingAxis_1" xlink:to="us-gaap_VestingDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_VestingAxis_1 To us-gaap_VestingDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis_1" xlink:title="us-gaap_IncomeStatementLocationAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain_1" xlink:title="us-gaap_IncomeStatementLocationDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_IncomeStatementLocationAxis_1" xlink:to="us-gaap_IncomeStatementLocationDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_IncomeStatementLocationAxis_1 To us-gaap_IncomeStatementLocationDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis_1" xlink:title="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain_1" xlink:title="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis_1" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis_1 To us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AchievementOfMilestoneAxis" xlink:label="bcyc_AchievementOfMilestoneAxis_1" xlink:title="bcyc_AchievementOfMilestoneAxis_1" />
    <link:loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AchievementOfMilestoneDomain" xlink:label="bcyc_AchievementOfMilestoneDomain_1" xlink:title="bcyc_AchievementOfMilestoneDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="bcyc_AchievementOfMilestoneAxis_1" xlink:to="bcyc_AchievementOfMilestoneDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:bcyc_AchievementOfMilestoneAxis_1 To bcyc_AchievementOfMilestoneDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_1" xlink:title="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_1" xlink:title="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_1" xlink:to="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_1 To us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductOrServiceAxis" xlink:label="srt_ProductOrServiceAxis_1" xlink:title="srt_ProductOrServiceAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain_1" xlink:title="srt_ProductsAndServicesDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_ProductOrServiceAxis_1" xlink:to="srt_ProductsAndServicesDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_ProductOrServiceAxis_1 To srt_ProductsAndServicesDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis_1" xlink:title="srt_CounterpartyNameAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain_1" xlink:title="srt_RepurchaseAgreementCounterpartyNameDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_CounterpartyNameAxis_1" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_CounterpartyNameAxis_1 To srt_RepurchaseAgreementCounterpartyNameDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedCostsOfUnprovedPropertiesExcludedFromAmortizationByPropertyOrProjectAxis" xlink:label="us-gaap_CapitalizedCostsOfUnprovedPropertiesExcludedFromAmortizationByPropertyOrProjectAxis_1" xlink:title="us-gaap_CapitalizedCostsOfUnprovedPropertiesExcludedFromAmortizationByPropertyOrProjectAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProjectMember" xlink:label="us-gaap_ProjectMember_1" xlink:title="us-gaap_ProjectMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_CapitalizedCostsOfUnprovedPropertiesExcludedFromAmortizationByPropertyOrProjectAxis_1" xlink:to="us-gaap_ProjectMember_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_CapitalizedCostsOfUnprovedPropertiesExcludedFromAmortizationByPropertyOrProjectAxis_1 To us-gaap_ProjectMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GainContingenciesByNatureAxis" xlink:label="us-gaap_GainContingenciesByNatureAxis_1" xlink:title="us-gaap_GainContingenciesByNatureAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GainContingencyNatureDomain" xlink:label="us-gaap_GainContingencyNatureDomain_1" xlink:title="us-gaap_GainContingencyNatureDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_GainContingenciesByNatureAxis_1" xlink:to="us-gaap_GainContingencyNatureDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_GainContingenciesByNatureAxis_1 To us-gaap_GainContingencyNatureDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_PerformanceObligationAxis" xlink:label="bcyc_PerformanceObligationAxis_1" xlink:title="bcyc_PerformanceObligationAxis_1" />
    <link:loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_PerformanceObligationDomain" xlink:label="bcyc_PerformanceObligationDomain_1" xlink:title="bcyc_PerformanceObligationDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="bcyc_PerformanceObligationAxis_1" xlink:to="bcyc_PerformanceObligationDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:bcyc_PerformanceObligationAxis_1 To bcyc_PerformanceObligationDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IndefiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="us-gaap_IndefiniteLivedIntangibleAssetsByMajorClassAxis_1" xlink:title="us-gaap_IndefiniteLivedIntangibleAssetsByMajorClassAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IndefiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_IndefiniteLivedIntangibleAssetsMajorClassNameDomain_1" xlink:title="us-gaap_IndefiniteLivedIntangibleAssetsMajorClassNameDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_IndefiniteLivedIntangibleAssetsByMajorClassAxis_1" xlink:to="us-gaap_IndefiniteLivedIntangibleAssetsMajorClassNameDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_IndefiniteLivedIntangibleAssetsByMajorClassAxis_1 To us-gaap_IndefiniteLivedIntangibleAssetsMajorClassNameDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationAxis" xlink:label="us-gaap_BalanceSheetLocationAxis_1" xlink:title="us-gaap_BalanceSheetLocationAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain" xlink:label="us-gaap_BalanceSheetLocationDomain_1" xlink:title="us-gaap_BalanceSheetLocationDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_BalanceSheetLocationAxis_1" xlink:to="us-gaap_BalanceSheetLocationDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_BalanceSheetLocationAxis_1 To us-gaap_BalanceSheetLocationDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxAuthorityAxis" xlink:label="us-gaap_IncomeTaxAuthorityAxis_1" xlink:title="us-gaap_IncomeTaxAuthorityAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain_1" xlink:title="us-gaap_IncomeTaxAuthorityDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_IncomeTaxAuthorityAxis_1" xlink:to="us-gaap_IncomeTaxAuthorityDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_IncomeTaxAuthorityAxis_1 To us-gaap_IncomeTaxAuthorityDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1" xlink:title="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_1" xlink:title="us-gaap_RelatedPartyDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1" xlink:to="us-gaap_RelatedPartyDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1 To us-gaap_RelatedPartyDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RelatedPartyTransactionAxis" xlink:label="us-gaap_RelatedPartyTransactionAxis_1" xlink:title="us-gaap_RelatedPartyTransactionAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain_1" xlink:title="us-gaap_RelatedPartyTransactionDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_RelatedPartyTransactionAxis_1" xlink:to="us-gaap_RelatedPartyTransactionDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_RelatedPartyTransactionAxis_1 To us-gaap_RelatedPartyTransactionDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_1" xlink:title="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain_1" xlink:title="us-gaap_AntidilutiveSecuritiesNameDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_1" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_1 To us-gaap_AntidilutiveSecuritiesNameDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetirementPlanSponsorLocationAxis" xlink:label="us-gaap_RetirementPlanSponsorLocationAxis_1" xlink:title="us-gaap_RetirementPlanSponsorLocationAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetirementPlanSponsorLocationDomain" xlink:label="us-gaap_RetirementPlanSponsorLocationDomain_1" xlink:title="us-gaap_RetirementPlanSponsorLocationDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_RetirementPlanSponsorLocationAxis_1" xlink:to="us-gaap_RetirementPlanSponsorLocationDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_RetirementPlanSponsorLocationAxis_1 To us-gaap_RetirementPlanSponsorLocationDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetirementPlanNameAxis" xlink:label="us-gaap_RetirementPlanNameAxis_1" xlink:title="us-gaap_RetirementPlanNameAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetirementPlanNameDomain" xlink:label="us-gaap_RetirementPlanNameDomain_1" xlink:title="us-gaap_RetirementPlanNameDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_RetirementPlanNameAxis_1" xlink:to="us-gaap_RetirementPlanNameDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_RetirementPlanNameAxis_1 To us-gaap_RetirementPlanNameDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis_1" xlink:title="us-gaap_FinancialInstrumentAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1" xlink:title="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_FinancialInstrumentAxis_1" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_FinancialInstrumentAxis_1 To us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RestatementAxis" xlink:label="srt_RestatementAxis_1" xlink:title="srt_RestatementAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RestatementDomain" xlink:label="srt_RestatementDomain_1" xlink:title="srt_RestatementDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_RestatementAxis_1" xlink:to="srt_RestatementDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_RestatementAxis_1 To srt_RestatementDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis_1" xlink:title="us-gaap_ClassOfWarrantOrRightAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_1" xlink:title="us-gaap_ClassOfWarrantOrRightDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_ClassOfWarrantOrRightAxis_1 To us-gaap_ClassOfWarrantOrRightDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:label="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis_1" xlink:title="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfAdoptionMember" xlink:label="us-gaap_TypeOfAdoptionMember_1" xlink:title="us-gaap_TypeOfAdoptionMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis_1" xlink:to="us-gaap_TypeOfAdoptionMember_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_AdjustmentsForNewAccountingPronouncementsAxis_1 To us-gaap_TypeOfAdoptionMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="us-gaap_MeasurementInputTypeAxis_1" xlink:title="us-gaap_MeasurementInputTypeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain_1" xlink:title="us-gaap_MeasurementInputTypeDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_MeasurementInputTypeAxis_1" xlink:to="us-gaap_MeasurementInputTypeDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_MeasurementInputTypeAxis_1 To us-gaap_MeasurementInputTypeDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TaxCreditCarryforwardAxis" xlink:label="us-gaap_TaxCreditCarryforwardAxis_1" xlink:title="us-gaap_TaxCreditCarryforwardAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TaxCreditCarryforwardNameDomain" xlink:label="us-gaap_TaxCreditCarryforwardNameDomain_1" xlink:title="us-gaap_TaxCreditCarryforwardNameDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_TaxCreditCarryforwardAxis_1" xlink:to="us-gaap_TaxCreditCarryforwardNameDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_TaxCreditCarryforwardAxis_1 To us-gaap_TaxCreditCarryforwardNameDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ShareRepurchaseProgramAxis" xlink:label="srt_ShareRepurchaseProgramAxis_1" xlink:title="us-gaap_ShareRepurchaseProgramAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ShareRepurchaseProgramDomain" xlink:label="srt_ShareRepurchaseProgramDomain_1" xlink:title="us-gaap_ShareRepurchaseProgramDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_ShareRepurchaseProgramAxis_1" xlink:to="srt_ShareRepurchaseProgramDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_ShareRepurchaseProgramAxis_1 To us-gaap_ShareRepurchaseProgramDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:label="us-gaap_StatementBusinessSegmentsAxis_1" xlink:title="us-gaap_StatementBusinessSegmentsAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain_1" xlink:title="us-gaap_SegmentDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_StatementBusinessSegmentsAxis_1" xlink:to="us-gaap_SegmentDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_StatementBusinessSegmentsAxis_1 To us-gaap_SegmentDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashAndCashEquivalentsAxis" xlink:label="us-gaap_CashAndCashEquivalentsAxis_1" xlink:title="us-gaap_CashAndCashEquivalentsAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_1" xlink:title="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_CashAndCashEquivalentsAxis_1" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_CashAndCashEquivalentsAxis_1 To us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringPlanAxis" xlink:label="us-gaap_RestructuringPlanAxis_1" xlink:title="us-gaap_RestructuringPlanAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringPlanDomain" xlink:label="us-gaap_RestructuringPlanDomain_1" xlink:title="us-gaap_RestructuringPlanDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="us-gaap_RestructuringPlanAxis_1" xlink:to="us-gaap_RestructuringPlanDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:us-gaap_RestructuringPlanAxis_1 To us-gaap_RestructuringPlanDomain_1" order="1" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://xbrl.sec.gov/ecd/role/IndividualsOnly" xlink:type="extended" xlink:title="Individuals">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/ecd/2025/ecd-2025.xsd#ecd_AllIndividualsMember" xlink:label="ecd_AllIndividualsMember" xlink:title="ecd_AllIndividualsMember" />
    <link:loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AlethiaYoungMember" xlink:label="bcyc_AlethiaYoungMember" xlink:title="bcyc_AlethiaYoungMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ecd_AllIndividualsMember" xlink:to="bcyc_AlethiaYoungMember" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:ecd_AllIndividualsMember To bcyc_AlethiaYoungMember" order="1" priority="0" />
    <link:loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CharlesSwantonMember" xlink:label="bcyc_CharlesSwantonMember" xlink:title="bcyc_CharlesSwantonMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ecd_AllIndividualsMember" xlink:to="bcyc_CharlesSwantonMember" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:title="definition:ecd_AllIndividualsMember To bcyc_CharlesSwantonMember" order="5" priority="0" />
  </link:definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DocumentDocumentAndEntityInformation" xlink:type="extended" xlink:title="995200090 - Document - Document and Entity Information" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentInformationTable" xlink:label="dei_DocumentInformationTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="dei_DocumentInformationTable" xlink:to="us-gaap_StatementClassOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_CommonStockMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AdrMember" xlink:label="dei_AdrMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="dei_AdrMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NonVotingOrdinarySharesMember" xlink:label="bcyc_NonVotingOrdinarySharesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="bcyc_NonVotingOrdinarySharesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentInformationLineItems" xlink:label="dei_DocumentInformationLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentInformationTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentType" xlink:label="dei_DocumentType" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentType" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentAnnualReport" xlink:label="dei_DocumentAnnualReport" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentAnnualReport" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentPeriodEndDate" xlink:label="dei_DocumentPeriodEndDate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentPeriodEndDate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentTransitionReport" xlink:label="dei_DocumentTransitionReport" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentTransitionReport" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityFileNumber" xlink:label="dei_EntityFileNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFileNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityRegistrantName" xlink:label="dei_EntityRegistrantName" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityRegistrantName" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityIncorporationStateCountryCode" xlink:label="dei_EntityIncorporationStateCountryCode" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityIncorporationStateCountryCode" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityTaxIdentificationNumber" xlink:label="dei_EntityTaxIdentificationNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityTaxIdentificationNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressAddressLine1" xlink:label="dei_EntityAddressAddressLine1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressAddressLine1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressCityOrTown" xlink:label="dei_EntityAddressCityOrTown" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressCityOrTown" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressCountry" xlink:label="dei_EntityAddressCountry" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressCountry" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressPostalZipCode" xlink:label="dei_EntityAddressPostalZipCode" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressPostalZipCode" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_CityAreaCode" xlink:label="dei_CityAreaCode" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CityAreaCode" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_LocalPhoneNumber" xlink:label="dei_LocalPhoneNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="15" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_LocalPhoneNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_Security12bTitle" xlink:label="dei_Security12bTitle" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="16" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_Security12bTitle" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_NoTradingSymbolFlag" xlink:label="dei_NoTradingSymbolFlag" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="17" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_NoTradingSymbolFlag" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_TradingSymbol" xlink:label="dei_TradingSymbol" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="18" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_TradingSymbol" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_SecurityExchangeName" xlink:label="dei_SecurityExchangeName" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="19" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_SecurityExchangeName" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityWellKnownSeasonedIssuer" xlink:label="dei_EntityWellKnownSeasonedIssuer" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="20" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityWellKnownSeasonedIssuer" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityVoluntaryFilers" xlink:label="dei_EntityVoluntaryFilers" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="21" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityVoluntaryFilers" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityCurrentReportingStatus" xlink:label="dei_EntityCurrentReportingStatus" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="22" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCurrentReportingStatus" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityInteractiveDataCurrent" xlink:label="dei_EntityInteractiveDataCurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="23" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityInteractiveDataCurrent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityFilerCategory" xlink:label="dei_EntityFilerCategory" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="24" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFilerCategory" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntitySmallBusiness" xlink:label="dei_EntitySmallBusiness" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="25" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntitySmallBusiness" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityEmergingGrowthCompany" xlink:label="dei_EntityEmergingGrowthCompany" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="26" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityEmergingGrowthCompany" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_IcfrAuditorAttestationFlag" xlink:label="dei_IcfrAuditorAttestationFlag" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="27" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_IcfrAuditorAttestationFlag" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentFinStmtErrorCorrectionFlag" xlink:label="dei_DocumentFinStmtErrorCorrectionFlag" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="28" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFinStmtErrorCorrectionFlag" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityShellCompany" xlink:label="dei_EntityShellCompany" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="29" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityShellCompany" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityPublicFloat" xlink:label="dei_EntityPublicFloat" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="30" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityPublicFloat" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityCommonStockSharesOutstanding" xlink:label="dei_EntityCommonStockSharesOutstanding" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="31" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCommonStockSharesOutstanding" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityCentralIndexKey" xlink:label="dei_EntityCentralIndexKey" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="32" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCentralIndexKey" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_CurrentFiscalYearEndDate" xlink:label="dei_CurrentFiscalYearEndDate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="33" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CurrentFiscalYearEndDate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentFiscalYearFocus" xlink:label="dei_DocumentFiscalYearFocus" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="34" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalYearFocus" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentFiscalPeriodFocus" xlink:label="dei_DocumentFiscalPeriodFocus" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="35" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalPeriodFocus" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AmendmentFlag" xlink:label="dei_AmendmentFlag" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="36" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AmendmentFlag" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AuditorName" xlink:label="dei_AuditorName" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="37" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AuditorName" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AuditorLocation" xlink:label="dei_AuditorLocation" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="38" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AuditorLocation" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AuditorFirmId" xlink:label="dei_AuditorFirmId" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="39" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AuditorFirmId" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/StatementConsolidatedStatementsOfShareholdersEquity" xlink:type="extended" xlink:title="995200300 - Statement - Consolidated Statements of Shareholders' Equity" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="us-gaap_AdditionalPaidInCapitalMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:label="us-gaap_AccumulatedOtherComprehensiveIncomeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AccumulatedOtherComprehensiveIncomeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsMember" xlink:label="us-gaap_RetainedEarningsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisSharePurchaseAgreementMember" xlink:label="bcyc_IonisSharePurchaseAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="bcyc_IonisSharePurchaseAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:label="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockholdersEquity" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="us-gaap_CommonStockSharesOutstanding" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_CommonStockSharesOutstanding" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" xlink:label="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" xlink:label="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent" xlink:label="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_NetIncomeLoss" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsTables" xlink:type="extended" xlink:title="99930903 - Disclosure - Significant agreements (Tables)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_BayerMember" xlink:label="bcyc_BayerMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_BayerMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTableTextBlock" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTableTextBlock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTableTextBlock" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_RevenuePerformanceObligationAllocationOfTransactionPriceTableTextBlock" xlink:label="bcyc_RevenuePerformanceObligationAllocationOfTransactionPriceTableTextBlock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_RevenuePerformanceObligationAllocationOfTransactionPriceTableTextBlock" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerAssetAndLiabilityTableTextBlock" xlink:label="us-gaap_ContractWithCustomerAssetAndLiabilityTableTextBlock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ContractWithCustomerAssetAndLiabilityTableTextBlock" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_RevenueChangeInContractWithCustomerAssetAndLiabilityTableTextBlock" xlink:label="bcyc_RevenueChangeInContractWithCustomerAssetAndLiabilityTableTextBlock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_RevenueChangeInContractWithCustomerAssetAndLiabilityTableTextBlock" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureNatureOfBusinessAndBasisOfPresentationShareActivityDetails" xlink:type="extended" xlink:title="99940102 - Disclosure - Nature of the business and basis of presentation - Share activity (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_InvestorsPurchaseAgreementMember" xlink:label="bcyc_InvestorsPurchaseAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="bcyc_InvestorsPurchaseAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_UnderwrittenPublicOfferingMember" xlink:label="bcyc_UnderwrittenPublicOfferingMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="bcyc_UnderwrittenPublicOfferingMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OverAllotmentOptionMember" xlink:label="us-gaap_OverAllotmentOptionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="bcyc_UnderwrittenPublicOfferingMember" xlink:to="us-gaap_OverAllotmentOptionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AtMarketOfferingMember" xlink:label="bcyc_AtMarketOfferingMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="bcyc_AtMarketOfferingMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_StatementClassOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AdrMember" xlink:label="dei_AdrMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="dei_AdrMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NonVotingOrdinarySharesMember" xlink:label="bcyc_NonVotingOrdinarySharesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="bcyc_NonVotingOrdinarySharesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_CommonStockSharesIssued" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_SharePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PaymentsOfStockIssuanceCosts" xlink:label="us-gaap_PaymentsOfStockIssuanceCosts" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_PaymentsOfStockIssuanceCosts" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSummaryOfSignificantAccountingPoliciesPropertyAndEquipmentGeographicDetails" xlink:type="extended" xlink:title="99940204 - Disclosure - Summary of significant accounting policies - Property and equipment, Geographic (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:label="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="srt_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquipmentMember" xlink:label="us-gaap_EquipmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_EquipmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ComputerEquipmentMember" xlink:label="us-gaap_ComputerEquipmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_ComputerEquipmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FurnitureAndFixturesMember" xlink:label="us-gaap_FurnitureAndFixturesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_FurnitureAndFixturesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:label="us-gaap_PropertyPlantAndEquipmentLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentUsefulLife" xlink:label="us-gaap_PropertyPlantAndEquipmentUsefulLife" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentUsefulLife" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfGeographicRegionsInWhichOperating" xlink:label="bcyc_NumberOfGeographicRegionsInWhichOperating" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="bcyc_NumberOfGeographicRegionsInWhichOperating" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSummaryOfSignificantAccountingPoliciesResearchAndDevelopmentIncentivesAndReceivableDetails" xlink:type="extended" xlink:title="99940205 - Disclosure - Summary of significant accounting policies - Research and development incentives and receivable (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementScenarioAxis" xlink:label="srt_StatementScenarioAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="srt_StatementScenarioAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ScenarioUnspecifiedDomain" xlink:label="srt_ScenarioUnspecifiedDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_StatementScenarioAxis" xlink:to="srt_ScenarioUnspecifiedDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CertainResearchAndDevelopmentExpendituresIncurredThereafterApril12023Member" xlink:label="bcyc_CertainResearchAndDevelopmentExpendituresIncurredThereafterApril12023Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_ScenarioUnspecifiedDomain" xlink:to="bcyc_CertainResearchAndDevelopmentExpendituresIncurredThereafterApril12023Member" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CertainResearchAndDevelopmentExpendituresIncurredThereafterApril12024Member" xlink:label="bcyc_CertainResearchAndDevelopmentExpendituresIncurredThereafterApril12024Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_ScenarioUnspecifiedDomain" xlink:to="bcyc_CertainResearchAndDevelopmentExpendituresIncurredThereafterApril12024Member" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:label="us-gaap_ResearchAndDevelopmentExpenseAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ResearchAndDevelopmentExpenseAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ResearchAndDevelopmentTaxReliefsProgramPercentageOfTaxCreditOnSurrenderableLosses" xlink:label="bcyc_ResearchAndDevelopmentTaxReliefsProgramPercentageOfTaxCreditOnSurrenderableLosses" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_ResearchAndDevelopmentTaxReliefsProgramPercentageOfTaxCreditOnSurrenderableLosses" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ResearchAndDevelopmentPercentageOfTaxCreditOnTotalExpenditure" xlink:label="bcyc_ResearchAndDevelopmentPercentageOfTaxCreditOnTotalExpenditure" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_ResearchAndDevelopmentPercentageOfTaxCreditOnTotalExpenditure" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ReductionOfResearchAndDevelopmentExpense" xlink:label="bcyc_ReductionOfResearchAndDevelopmentExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_ReductionOfResearchAndDevelopmentExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ResearchAndDevelopmentIncentivesReceivableCurrent" xlink:label="bcyc_ResearchAndDevelopmentIncentivesReceivableCurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_ResearchAndDevelopmentIncentivesReceivableCurrent" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureFairValueOfFinancialAssetsAndLiabilitiesFinancialAssetsAndLiabilitiesMeasuredAtFairValueDetails" xlink:type="extended" xlink:title="99940301 - Disclosure - Fair value of financial assets and liabilities - Financial assets and liabilities measured at fair value (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByMeasurementFrequencyAxis" xlink:label="us-gaap_FairValueByMeasurementFrequencyAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByMeasurementFrequencyAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsRecurringMember" xlink:label="us-gaap_FairValueMeasurementsRecurringMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FairValueMeasurementFrequencyDomain" xlink:to="us-gaap_FairValueMeasurementsRecurringMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashAndCashEquivalentsAxis" xlink:label="us-gaap_CashAndCashEquivalentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_CashAndCashEquivalentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashEquivalentsMember" xlink:label="us-gaap_CashEquivalentsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:to="us-gaap_CashEquivalentsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MoneyMarketFundsAtCarryingValue" xlink:label="us-gaap_MoneyMarketFundsAtCarryingValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_MoneyMarketFundsAtCarryingValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialLiabilitiesFairValueDisclosure" xlink:label="us-gaap_FinancialLiabilitiesFairValueDisclosure" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_FinancialLiabilitiesFairValueDisclosure" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosurePropertyAndEquipmentNetDetails" xlink:type="extended" xlink:title="99940401 - Disclosure - Property and equipment, net (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:label="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquipmentMember" xlink:label="us-gaap_EquipmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_EquipmentMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LeaseholdImprovementsMember" xlink:label="us-gaap_LeaseholdImprovementsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_LeaseholdImprovementsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ComputerEquipmentMember" xlink:label="us-gaap_ComputerEquipmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_ComputerEquipmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FurnitureAndFixturesMember" xlink:label="us-gaap_FurnitureAndFixturesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_FurnitureAndFixturesMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:label="us-gaap_PropertyPlantAndEquipmentLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentGross" xlink:label="us-gaap_PropertyPlantAndEquipmentGross" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentGross" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseRightOfUseAssetBeforeAccumulatedAmortization" xlink:label="us-gaap_FinanceLeaseRightOfUseAssetBeforeAccumulatedAmortization" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_FinanceLeaseRightOfUseAssetBeforeAccumulatedAmortization" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentAndFinanceLeaseRightOfUseAssetBeforeAccumulatedDepreciationAndAmortization" xlink:label="us-gaap_PropertyPlantAndEquipmentAndFinanceLeaseRightOfUseAssetBeforeAccumulatedDepreciationAndAmortization" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentAndFinanceLeaseRightOfUseAssetBeforeAccumulatedDepreciationAndAmortization" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentAndFinanceLeaseRightOfUseAssetAccumulatedDepreciationAndAmortization" xlink:label="us-gaap_PropertyPlantAndEquipmentAndFinanceLeaseRightOfUseAssetAccumulatedDepreciationAndAmortization" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentAndFinanceLeaseRightOfUseAssetAccumulatedDepreciationAndAmortization" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentAndFinanceLeaseRightOfUseAssetAfterAccumulatedDepreciationAndAmortization" xlink:label="us-gaap_PropertyPlantAndEquipmentAndFinanceLeaseRightOfUseAssetAfterAccumulatedDepreciationAndAmortization" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentAndFinanceLeaseRightOfUseAssetAfterAccumulatedDepreciationAndAmortization" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DepreciationAndAmortization" xlink:label="us-gaap_DepreciationAndAmortization" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_DepreciationAndAmortization" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseRightOfUseAssetAmortization" xlink:label="us-gaap_FinanceLeaseRightOfUseAssetAmortization" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_FinanceLeaseRightOfUseAssetAmortization" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureAccruedExpensesAndOtherCurrentLiabilitiesAdditionalInformationDetails" xlink:type="extended" xlink:title="99940502 - Disclosure - Accrued expenses and other current liabilities - Additional information (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfRestructuringAndRelatedCostsTable" xlink:label="us-gaap_ScheduleOfRestructuringAndRelatedCostsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringPlanAxis" xlink:label="us-gaap_RestructuringPlanAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfRestructuringAndRelatedCostsTable" xlink:to="us-gaap_RestructuringPlanAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringPlanDomain" xlink:label="us-gaap_RestructuringPlanDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RestructuringPlanAxis" xlink:to="us-gaap_RestructuringPlanDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CostReductionInitiativesMember" xlink:label="bcyc_CostReductionInitiativesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RestructuringPlanDomain" xlink:to="bcyc_CostReductionInitiativesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_DiscoveryResearchActivitiesConsolidationMember" xlink:label="bcyc_DiscoveryResearchActivitiesConsolidationMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RestructuringPlanDomain" xlink:to="bcyc_DiscoveryResearchActivitiesConsolidationMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfRestructuringAndRelatedCostsTable" xlink:to="us-gaap_IncomeStatementLocationAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseMember" xlink:label="us-gaap_ResearchAndDevelopmentExpenseMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_ResearchAndDevelopmentExpenseMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringCostAndReserveLineItems" xlink:label="us-gaap_RestructuringCostAndReserveLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_RestructuringCostAndReserveLineItems" xlink:to="us-gaap_ScheduleOfRestructuringAndRelatedCostsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringAndRelatedCostNumberOfPositionsEliminatedPeriodPercent" xlink:label="us-gaap_RestructuringAndRelatedCostNumberOfPositionsEliminatedPeriodPercent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RestructuringCostAndReserveLineItems" xlink:to="us-gaap_RestructuringAndRelatedCostNumberOfPositionsEliminatedPeriodPercent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringAndRelatedCostExpectedCost1" xlink:label="us-gaap_RestructuringAndRelatedCostExpectedCost1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_RestructuringCostAndReserveLineItems" xlink:to="us-gaap_RestructuringAndRelatedCostExpectedCost1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringAndRelatedCostIncurredCost" xlink:label="us-gaap_RestructuringAndRelatedCostIncurredCost" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_RestructuringCostAndReserveLineItems" xlink:to="us-gaap_RestructuringAndRelatedCostIncurredCost" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PaymentsForRestructuring" xlink:label="us-gaap_PaymentsForRestructuring" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_RestructuringCostAndReserveLineItems" xlink:to="us-gaap_PaymentsForRestructuring" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringReserveCurrent" xlink:label="us-gaap_RestructuringReserveCurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_RestructuringCostAndReserveLineItems" xlink:to="us-gaap_RestructuringReserveCurrent" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureDebtDetails" xlink:type="extended" xlink:title="99940601 - Disclosure - Debt (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable" xlink:label="us-gaap_DebtInstrumentTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_LoanAndSecurityAgreementMember" xlink:label="bcyc_LoanAndSecurityAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_LoanAndSecurityAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis" xlink:label="us-gaap_LongtermDebtTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_LongtermDebtTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_LongtermDebtTypeAxis" xlink:to="us-gaap_LongtermDebtTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_TermLoanMember" xlink:label="bcyc_TermLoanMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_LongtermDebtTypeDomain" xlink:to="bcyc_TermLoanMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_InitialTermLoanMember" xlink:label="bcyc_InitialTermLoanMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="bcyc_TermLoanMember" xlink:to="bcyc_InitialTermLoanMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="srt_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems" xlink:label="us-gaap_DebtInstrumentLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" xlink:label="us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="us-gaap_DebtInstrumentFaceAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentFaceAmount" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" xlink:label="us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="us-gaap_DebtInstrumentInterestRateStatedPercentage" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentInterestRateStatedPercentage" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_DebtInstrumentEndOfTermChargeRate" xlink:label="bcyc_DebtInstrumentEndOfTermChargeRate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="bcyc_DebtInstrumentEndOfTermChargeRate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InterestPayableCurrent" xlink:label="us-gaap_InterestPayableCurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_InterestPayableCurrent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_DebtEndOfTermCharge" xlink:label="bcyc_DebtEndOfTermCharge" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="bcyc_DebtEndOfTermCharge" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_DebtInstrumentPrepaymentCharges" xlink:label="bcyc_DebtInstrumentPrepaymentCharges" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="bcyc_DebtInstrumentPrepaymentCharges" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RepaymentsOfLongTermDebt" xlink:label="us-gaap_RepaymentsOfLongTermDebt" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_RepaymentsOfLongTermDebt" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GainsLossesOnExtinguishmentOfDebt" xlink:label="us-gaap_GainsLossesOnExtinguishmentOfDebt" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_GainsLossesOnExtinguishmentOfDebt" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InterestExpenseLongTermDebt" xlink:label="us-gaap_InterestExpenseLongTermDebt" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_InterestExpenseLongTermDebt" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureOrdinarySharesDetails" xlink:type="extended" xlink:title="99940701 - Disclosure - Ordinary shares (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:label="us-gaap_ScheduleOfStockByClassTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_StatementClassOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NonVotingOrdinarySharesMember" xlink:label="bcyc_NonVotingOrdinarySharesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="bcyc_NonVotingOrdinarySharesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AdrMember" xlink:label="dei_AdrMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="dei_AdrMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_VotingOrdinarySharesMember" xlink:label="bcyc_VotingOrdinarySharesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="bcyc_VotingOrdinarySharesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_StatementEquityComponentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockLineItems" xlink:label="us-gaap_ClassOfStockLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfVotePerShare" xlink:label="bcyc_NumberOfVotePerShare" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="bcyc_NumberOfVotePerShare" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockDividendsPerShareDeclared" xlink:label="us-gaap_CommonStockDividendsPerShareDeclared" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockDividendsPerShareDeclared" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockSharesIssued" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="us-gaap_CommonStockSharesOutstanding" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockSharesOutstanding" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationDetails" xlink:type="extended" xlink:title="99940801 - Disclosure - Share-based compensation (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_PlanNameAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareOptionPlan2020Member" xlink:label="bcyc_ShareOptionPlan2020Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PlanNameDomain" xlink:to="bcyc_ShareOptionPlan2020Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareOptionPlan2019Member" xlink:label="bcyc_ShareOptionPlan2019Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PlanNameDomain" xlink:to="bcyc_ShareOptionPlan2019Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_InducementPlan2024Member" xlink:label="bcyc_InducementPlan2024Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PlanNameDomain" xlink:to="bcyc_InducementPlan2024Member" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_PreIpoShareOptionsAndRestrictedSharesMember" xlink:label="bcyc_PreIpoShareOptionsAndRestrictedSharesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PlanNameDomain" xlink:to="bcyc_PreIpoShareOptionsAndRestrictedSharesMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="srt_TitleOfIndividualAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_DirectorMember" xlink:label="srt_DirectorMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:to="srt_DirectorMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="srt_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="4" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockCompensationPlanMember" xlink:label="us-gaap_StockCompensationPlanMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_StockCompensationPlanMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedPaymentArrangementThreeYearVestMember" xlink:label="bcyc_ShareBasedPaymentArrangementThreeYearVestMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_StockCompensationPlanMember" xlink:to="bcyc_ShareBasedPaymentArrangementThreeYearVestMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockMember" xlink:label="us-gaap_RestrictedStockMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember" xlink:label="us-gaap_EmployeeStockMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GranteeStatusAxis" xlink:label="us-gaap_GranteeStatusAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="5" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_GranteeStatusAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:label="us-gaap_ShareBasedPaymentArrangementEmployeeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="us-gaap_ShareBasedPaymentArrangementEmployeeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingAxis" xlink:label="us-gaap_VestingAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="6" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_VestingAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheOneMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheOneMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationAwardTrancheTwoMember" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheTwoMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheTwoMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardsShareConversionRatio" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardsShareConversionRatio" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardsShareConversionRatio" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementsByShareBasedPaymentAwardVestingNumberOfEqualMonthlyInstallments" xlink:label="bcyc_ShareBasedCompensationArrangementsByShareBasedPaymentAwardVestingNumberOfEqualMonthlyInstallments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="bcyc_ShareBasedCompensationArrangementsByShareBasedPaymentAwardVestingNumberOfEqualMonthlyInstallments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAuthorized" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAuthorized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAuthorized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:label="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardAnnualIncreasePercentage" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardAnnualIncreasePercentage" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardAnnualIncreasePercentage" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardRequisiteServicePeriod1" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardRequisiteServicePeriod1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardRequisiteServicePeriod1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementsByShareBasedPaymentAwardVestingNumberOfEqualQuarterlyInstallments" xlink:label="bcyc_ShareBasedCompensationArrangementsByShareBasedPaymentAwardVestingNumberOfEqualQuarterlyInstallments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="bcyc_ShareBasedCompensationArrangementsByShareBasedPaymentAwardVestingNumberOfEqualQuarterlyInstallments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementsByShareBasedPaymentAwardVestingNumberOfEqualAnnualInstallments" xlink:label="bcyc_ShareBasedCompensationArrangementsByShareBasedPaymentAwardVestingNumberOfEqualAnnualInstallments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="bcyc_ShareBasedCompensationArrangementsByShareBasedPaymentAwardVestingNumberOfEqualAnnualInstallments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfQuarterlyInstallments" xlink:label="bcyc_NumberOfQuarterlyInstallments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="15" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="bcyc_NumberOfQuarterlyInstallments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="16" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationShareBasedCompensationExpenseDetails" xlink:type="extended" xlink:title="99940802 - Disclosure - Share-based compensation - Share based compensation expense (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_IncomeStatementLocationAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseMember" xlink:label="us-gaap_ResearchAndDevelopmentExpenseMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_ResearchAndDevelopmentExpenseMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationShareOptionsDetails" xlink:type="extended" xlink:title="99940803 - Disclosure - Share-based compensation - Share options (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockCompensationPlanMember" xlink:label="us-gaap_StockCompensationPlanMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_StockCompensationPlanMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageContractualTermAbstract" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageContractualTermAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageContractualTermAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageContractualTermAbstract" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageRemainingContractualTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageRemainingContractualTerm1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageContractualTermAbstract" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageRemainingContractualTerm1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableWeightedAverageRemainingContractualTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableWeightedAverageRemainingContractualTerm1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageContractualTermAbstract" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableWeightedAverageRemainingContractualTerm1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingAggregateIntrinsicValueAbstract" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingAggregateIntrinsicValueAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingAggregateIntrinsicValueAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingAggregateIntrinsicValueAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingAggregateIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingAggregateIntrinsicValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingAggregateIntrinsicValueAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingAggregateIntrinsicValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingAggregateIntrinsicValueAbstract" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationAssumptionsUsedInBlackScholesOptionPricingModelToDetermineFairValueOfShareOptionsDetails" xlink:type="extended" xlink:title="99940804 - Disclosure - Share-based compensation - Assumptions used in the Black Scholes option pricing model to determine the fair value of share options (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockCompensationPlanMember" xlink:label="us-gaap_StockCompensationPlanMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_StockCompensationPlanMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="srt_TitleOfIndividualAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_EmployeeAndDirectorMember" xlink:label="bcyc_EmployeeAndDirectorMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:to="bcyc_EmployeeAndDirectorMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedAbstract" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedAbstract" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardRequisiteServicePeriod1" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardRequisiteServicePeriod1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardRequisiteServicePeriod1" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureShareBasedCompensationRestrictedShareUnitsDetails" xlink:type="extended" xlink:title="99940805 - Disclosure - Share-based compensation - Restricted share units (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockMember" xlink:label="us-gaap_RestrictedStockMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_PlanNameAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareOptionPlan2020Member" xlink:label="bcyc_ShareOptionPlan2020Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PlanNameDomain" xlink:to="bcyc_ShareOptionPlan2020Member" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GranteeStatusAxis" xlink:label="us-gaap_GranteeStatusAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_GranteeStatusAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedPaymentArrangementNonemployeeMember" xlink:label="us-gaap_ShareBasedPaymentArrangementNonemployeeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="us-gaap_ShareBasedPaymentArrangementNonemployeeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="4" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="srt_TitleOfIndividualAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_DirectorMember" xlink:label="srt_DirectorMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:to="srt_DirectorMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndSettledInPeriod" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndSettledInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndSettledInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndDeferredInPeriod" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndDeferredInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndDeferredInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodSubjectToDeferredSettlement" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodSubjectToDeferredSettlement" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodSubjectToDeferredSettlement" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsOutstandingNumber" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsOutstandingNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsOutstandingNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndSettledInPeriodWeightedAverageGrantDateFairValue" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndSettledInPeriodWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndSettledInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndDeferredInPeriodWeightedAverageGrantDateFairValue" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndDeferredInPeriodWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedAndDeferredInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodSubjectToDeferredSettlementWeightedAverageGrantDateFairValue" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodSubjectToDeferredSettlementWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodSubjectToDeferredSettlementWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsOutstandingWeightedAverageGrantDateFairValue" xlink:label="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsOutstandingWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="bcyc_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsOutstandingWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodTotalFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodTotalFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodTotalFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsCollaborationRevenuesDetails" xlink:type="extended" xlink:title="99940901 - Disclosure - Significant agreements - Collaboration revenues (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_BayerMember" xlink:label="bcyc_BayerMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_BayerMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NovartisCollaborationAgreementMember" xlink:label="bcyc_NovartisCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_NovartisCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_GenentechMember" xlink:label="bcyc_GenentechMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_GenentechMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AstrazenecaCollaborationAgreementMember" xlink:label="bcyc_AstrazenecaCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_AstrazenecaCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisCollaborationAgreementMember" xlink:label="bcyc_IonisCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_IonisCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsBayerCollaborationAgreementDetails" xlink:type="extended" xlink:title="99940902 - Disclosure - Significant agreements - Bayer Collaboration Agreement (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_BayerMember" xlink:label="bcyc_BayerMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_BayerMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AchievementOfMilestoneAxis" xlink:label="bcyc_AchievementOfMilestoneAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="bcyc_AchievementOfMilestoneAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AchievementOfMilestoneDomain" xlink:label="bcyc_AchievementOfMilestoneDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="bcyc_AchievementOfMilestoneAxis" xlink:to="bcyc_AchievementOfMilestoneDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_DevelopmentAndRegulatoryMilestoneMember" xlink:label="bcyc_DevelopmentAndRegulatoryMilestoneMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="bcyc_AchievementOfMilestoneDomain" xlink:to="bcyc_DevelopmentAndRegulatoryMilestoneMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MilestoneAcrossAllPotentialTargetProgramsMember" xlink:label="bcyc_MilestoneAcrossAllPotentialTargetProgramsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="bcyc_AchievementOfMilestoneDomain" xlink:to="bcyc_MilestoneAcrossAllPotentialTargetProgramsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="srt_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAgreementNumberOfTargets" xlink:label="bcyc_CollaborationAgreementNumberOfTargets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAgreementNumberOfTargets" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerLiability" xlink:label="us-gaap_ContractWithCustomerLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ContractWithCustomerLiability" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaterialRightsExerciseOrExpiryTerm" xlink:label="bcyc_MaterialRightsExerciseOrExpiryTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_MaterialRightsExerciseOrExpiryTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAndLicenseAgreementNumberOfCandidateCompoundsIntoFurtherDevelopment" xlink:label="bcyc_CollaborationAndLicenseAgreementNumberOfCandidateCompoundsIntoFurtherDevelopment" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAndLicenseAgreementNumberOfCandidateCompoundsIntoFurtherDevelopment" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAgreementTieredSalesMilestonePaymentsReceivable" xlink:label="bcyc_CollaborationAgreementTieredSalesMilestonePaymentsReceivable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAgreementTieredSalesMilestonePaymentsReceivable" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAgreementSalesMilestonePaymentsReceivable" xlink:label="bcyc_CollaborationAgreementSalesMilestonePaymentsReceivable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAgreementSalesMilestonePaymentsReceivable" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_PeriodOverWhichPerformanceObligationsWillBePerformed" xlink:label="bcyc_PeriodOverWhichPerformanceObligationsWillBePerformed" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_PeriodOverWhichPerformanceObligationsWillBePerformed" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_EstimatedAmountOfReimbursementPayableToContractResearchOrganizations" xlink:label="bcyc_EstimatedAmountOfReimbursementPayableToContractResearchOrganizations" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_EstimatedAmountOfReimbursementPayableToContractResearchOrganizations" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfResearchProgramsNoticeOfTerminationReceived" xlink:label="bcyc_NumberOfResearchProgramsNoticeOfTerminationReceived" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfResearchProgramsNoticeOfTerminationReceived" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" xlink:label="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsBayerCollaborationAgreementAccountingAnalysisDetails" xlink:type="extended" xlink:title="99940903 - Disclosure - Significant agreements - Bayer Collaboration Agreement - Accounting Analysis (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_BayerMember" xlink:label="bcyc_BayerMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_BayerMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_TwoCombinedPerformanceObligationsRelatedToLicensesAndResearchAndDevelopmentServicesAssociatedWithRadiopharmaceuticalCompoundsDirectedToFirstAndSecondTargetsMember" xlink:label="bcyc_TwoCombinedPerformanceObligationsRelatedToLicensesAndResearchAndDevelopmentServicesAssociatedWithRadiopharmaceuticalCompoundsDirectedToFirstAndSecondTargetsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_TwoCombinedPerformanceObligationsRelatedToLicensesAndResearchAndDevelopmentServicesAssociatedWithRadiopharmaceuticalCompoundsDirectedToFirstAndSecondTargetsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaterialRightAssociatedWithLimitedSubstitutionRightsForEitherFirstOrSecondTargetMember" xlink:label="bcyc_MaterialRightAssociatedWithLimitedSubstitutionRightsForEitherFirstOrSecondTargetMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_MaterialRightAssociatedWithLimitedSubstitutionRightsForEitherFirstOrSecondTargetMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_TwoMaterialRightsAssociatedWithOptionToProgressRadiopharmaceuticalCandidatesDirectedToFirstAndSecondTargetsIntoFurtherDevelopmentMember" xlink:label="bcyc_TwoMaterialRightsAssociatedWithOptionToProgressRadiopharmaceuticalCandidatesDirectedToFirstAndSecondTargetsIntoFurtherDevelopmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_TwoMaterialRightsAssociatedWithOptionToProgressRadiopharmaceuticalCandidatesDirectedToFirstAndSecondTargetsIntoFurtherDevelopmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_TwoMaterialRightsAssociatedWithOptionToProgressNonRadiopharmaceuticalCompoundDirectedToFirstAndSecondTargetsMember" xlink:label="bcyc_TwoMaterialRightsAssociatedWithOptionToProgressNonRadiopharmaceuticalCompoundDirectedToFirstAndSecondTargetsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_TwoMaterialRightsAssociatedWithOptionToProgressNonRadiopharmaceuticalCompoundDirectedToFirstAndSecondTargetsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaterialRightForOptionToExpandCollaborationToIncludeThirdTargetAndUnderlyingAdditionalOptionRightsMember" xlink:label="bcyc_MaterialRightForOptionToExpandCollaborationToIncludeThirdTargetAndUnderlyingAdditionalOptionRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_MaterialRightForOptionToExpandCollaborationToIncludeThirdTargetAndUnderlyingAdditionalOptionRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfCombinedPerformanceObligations" xlink:label="bcyc_NumberOfCombinedPerformanceObligations" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfCombinedPerformanceObligations" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfMaterialRights" xlink:label="bcyc_NumberOfMaterialRights" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfMaterialRights" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" xlink:label="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsNovartisCollaborationAgreementDetails" xlink:type="extended" xlink:title="99940904 - Disclosure - Significant agreements - Novartis Collaboration Agreement (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NovartisCollaborationAgreementMember" xlink:label="bcyc_NovartisCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_NovartisCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_TwoMaterialRightsAssociatedWithLimitedSubstitutionRightsMember" xlink:label="bcyc_TwoMaterialRightsAssociatedWithLimitedSubstitutionRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_TwoMaterialRightsAssociatedWithLimitedSubstitutionRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_TwoMaterialRightsAssociatedWithOptionsToProgressDevelopmentCandidatesIncorporatingRadionuclidesMember" xlink:label="bcyc_TwoMaterialRightsAssociatedWithOptionsToProgressDevelopmentCandidatesIncorporatingRadionuclidesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_TwoMaterialRightsAssociatedWithOptionsToProgressDevelopmentCandidatesIncorporatingRadionuclidesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_TwoMaterialRightsAssociatedWithOptionsToProgressDevelopmentCandidatesNotIncorporatingRadionuclidesMember" xlink:label="bcyc_TwoMaterialRightsAssociatedWithOptionsToProgressDevelopmentCandidatesNotIncorporatingRadionuclidesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_TwoMaterialRightsAssociatedWithOptionsToProgressDevelopmentCandidatesNotIncorporatingRadionuclidesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ExpirationOfTwoMaterialRightsAssociateWithLimitedSubstitutionRightsMember" xlink:label="bcyc_ExpirationOfTwoMaterialRightsAssociateWithLimitedSubstitutionRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_ExpirationOfTwoMaterialRightsAssociateWithLimitedSubstitutionRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAgreementNumberOfTargets" xlink:label="bcyc_CollaborationAgreementNumberOfTargets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAgreementNumberOfTargets" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" xlink:label="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfCombinedPerformanceObligations" xlink:label="bcyc_NumberOfCombinedPerformanceObligations" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfCombinedPerformanceObligations" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfMaterialRights" xlink:label="bcyc_NumberOfMaterialRights" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfMaterialRights" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" xlink:label="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerLiability" xlink:label="us-gaap_ContractWithCustomerLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ContractWithCustomerLiability" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_EstimatedVariableConsiderationNoLongerConstrained" xlink:label="bcyc_EstimatedVariableConsiderationNoLongerConstrained" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_EstimatedVariableConsiderationNoLongerConstrained" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaterialRightsExerciseOrExpiryTerm" xlink:label="bcyc_MaterialRightsExerciseOrExpiryTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_MaterialRightsExerciseOrExpiryTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAgreementTieredSalesMilestonePaymentsReceivable" xlink:label="bcyc_CollaborationAgreementTieredSalesMilestonePaymentsReceivable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAgreementTieredSalesMilestonePaymentsReceivable" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAgreementSalesMilestonePaymentsReceivable" xlink:label="bcyc_CollaborationAgreementSalesMilestonePaymentsReceivable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAgreementSalesMilestonePaymentsReceivable" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_PeriodOverWhichPerformanceObligationsWillBePerformed" xlink:label="bcyc_PeriodOverWhichPerformanceObligationsWillBePerformed" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_PeriodOverWhichPerformanceObligationsWillBePerformed" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaximumDaysAllowedForCancellationOfContractsPriorWrittenNotice" xlink:label="bcyc_MaximumDaysAllowedForCancellationOfContractsPriorWrittenNotice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_MaximumDaysAllowedForCancellationOfContractsPriorWrittenNotice" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsIonisEvaluationAndOptionAgreementDetails" xlink:type="extended" xlink:title="99940906 - Disclosure - Significant agreements - Ionis Evaluation and Option Agreement (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisEvaluationAndOptionAgreementMember" xlink:label="bcyc_IonisEvaluationAndOptionAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_IonisEvaluationAndOptionAgreementMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" xlink:label="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsIonisCollaborationAgreementDetails" xlink:type="extended" xlink:title="99940907 - Disclosure - Significant agreements - Ionis Collaboration Agreement (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisEvaluationAndOptionAgreementMember" xlink:label="bcyc_IonisEvaluationAndOptionAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_IonisEvaluationAndOptionAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisCollaborationAgreementMember" xlink:label="bcyc_IonisCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_IonisCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_EstimatedAmountOfReimbursementPayableToContractResearchOrganizations" xlink:label="bcyc_EstimatedAmountOfReimbursementPayableToContractResearchOrganizations" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_EstimatedAmountOfReimbursementPayableToContractResearchOrganizations" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAgreementNumberOfTargets" xlink:label="bcyc_CollaborationAgreementNumberOfTargets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAgreementNumberOfTargets" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborativeResearchPlanResearchAndDiscoveryActivitiesTerm" xlink:label="bcyc_CollaborativeResearchPlanResearchAndDiscoveryActivitiesTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborativeResearchPlanResearchAndDiscoveryActivitiesTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" xlink:label="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfPaymentsOfMidSingleDigitMillionDollarFailingToAchieveSpecifiedDevelopmentDiligenceMilestone" xlink:label="bcyc_NumberOfPaymentsOfMidSingleDigitMillionDollarFailingToAchieveSpecifiedDevelopmentDiligenceMilestone" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfPaymentsOfMidSingleDigitMillionDollarFailingToAchieveSpecifiedDevelopmentDiligenceMilestone" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfYearsOverWhichRoyaltyIsPayable" xlink:label="bcyc_NumberOfYearsOverWhichRoyaltyIsPayable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfYearsOverWhichRoyaltyIsPayable" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsIonisSharePurchaseAgreementDetails" xlink:type="extended" xlink:title="99940908 - Disclosure - Significant agreements - Ionis Share Purchase Agreement (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisCollaborationAgreementMember" xlink:label="bcyc_IonisCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_IonisCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisSharePurchaseAgreementMember" xlink:label="bcyc_IonisSharePurchaseAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="bcyc_IonisSharePurchaseAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_StatementClassOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AdrMember" xlink:label="dei_AdrMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="dei_AdrMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_SharePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_FairValueOfSharesHeld" xlink:label="bcyc_FairValueOfSharesHeld" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_FairValueOfSharesHeld" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" xlink:label="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsIonisAccountingAnalysisDetails" xlink:type="extended" xlink:title="99940909 - Disclosure - Significant agreements - Ionis Accounting Analysis (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisCollaborationAgreementMember" xlink:label="bcyc_IonisCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_IonisCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisEvaluationAndOptionAgreementMember" xlink:label="bcyc_IonisEvaluationAndOptionAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_IonisEvaluationAndOptionAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AmendedIonisCollaborationAgreementMember" xlink:label="bcyc_AmendedIonisCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_AmendedIonisCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisSharePurchaseAgreementMember" xlink:label="bcyc_IonisSharePurchaseAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="bcyc_IonisSharePurchaseAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="srt_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_EstimatedAmountOfReimbursementPayableToContractResearchOrganizations" xlink:label="bcyc_EstimatedAmountOfReimbursementPayableToContractResearchOrganizations" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_EstimatedAmountOfReimbursementPayableToContractResearchOrganizations" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" xlink:label="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" xlink:label="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerLiability" xlink:label="us-gaap_ContractWithCustomerLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ContractWithCustomerLiability" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaterialRightsExerciseOrExpiryTerm" xlink:label="bcyc_MaterialRightsExerciseOrExpiryTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_MaterialRightsExerciseOrExpiryTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfMaterialRightsAssociatedWithFuturePayments" xlink:label="bcyc_NumberOfMaterialRightsAssociatedWithFuturePayments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfMaterialRightsAssociatedWithFuturePayments" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsGenentechCollaborationAgreementDetails" xlink:type="extended" xlink:title="99940910 - Disclosure - Significant agreements - Genentech Collaboration Agreement (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" priority="2" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_GenentechMember" xlink:label="bcyc_GenentechMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_GenentechMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" priority="2" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_SpecifiedTargetingArmMaterialRightArmProgramOneMember" xlink:label="bcyc_SpecifiedTargetingArmMaterialRightArmProgramOneMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_SpecifiedTargetingArmMaterialRightArmProgramOneMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_TwoMaterialRightsAssociatedWithLsrGoOptionForCollaborationProgramsOneAndTwoMember" xlink:label="bcyc_TwoMaterialRightsAssociatedWithLsrGoOptionForCollaborationProgramsOneAndTwoMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_TwoMaterialRightsAssociatedWithLsrGoOptionForCollaborationProgramsOneAndTwoMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaterialRightsAssociatedWithLsrGoOptionForCollaborationProgramsOneMember" xlink:label="bcyc_MaterialRightsAssociatedWithLsrGoOptionForCollaborationProgramsOneMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_MaterialRightsAssociatedWithLsrGoOptionForCollaborationProgramsOneMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationProgramThreePerformanceObligationMember" xlink:label="bcyc_CollaborationProgramThreePerformanceObligationMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_CollaborationProgramThreePerformanceObligationMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaterialRightsMember" xlink:label="bcyc_MaterialRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_MaterialRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="srt_RangeAxis" priority="2" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfPotentialDevelopmentCandidates" xlink:label="bcyc_NumberOfPotentialDevelopmentCandidates" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfPotentialDevelopmentCandidates" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerLiabilityRevenueRecognized" xlink:label="us-gaap_ContractWithCustomerLiabilityRevenueRecognized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ContractWithCustomerLiabilityRevenueRecognized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerLiability" xlink:label="us-gaap_ContractWithCustomerLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ContractWithCustomerLiability" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfImmunoOncologyTargets" xlink:label="bcyc_NumberOfImmunoOncologyTargets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfImmunoOncologyTargets" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AdditionalNumberOfImmunoOncologyTargets" xlink:label="bcyc_AdditionalNumberOfImmunoOncologyTargets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_AdditionalNumberOfImmunoOncologyTargets" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ExpansionFeePerAdditionalCollaborationProgram" xlink:label="bcyc_ExpansionFeePerAdditionalCollaborationProgram" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_ExpansionFeePerAdditionalCollaborationProgram" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfCollaborationPrograms" xlink:label="bcyc_NumberOfCollaborationPrograms" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfCollaborationPrograms" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfExpansionOptionCollaborationProgramsDevGo" xlink:label="bcyc_NumberOfExpansionOptionCollaborationProgramsDevGo" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfExpansionOptionCollaborationProgramsDevGo" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAgreementSalesMilestonePaymentsReceivable" xlink:label="bcyc_CollaborationAgreementSalesMilestonePaymentsReceivable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAgreementSalesMilestonePaymentsReceivable" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfYearsOverWhichRoyaltyIsPayable" xlink:label="bcyc_NumberOfYearsOverWhichRoyaltyIsPayable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_NumberOfYearsOverWhichRoyaltyIsPayable" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_PeriodOverWhichPerformanceObligationsWillBePerformed" xlink:label="bcyc_PeriodOverWhichPerformanceObligationsWillBePerformed" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_PeriodOverWhichPerformanceObligationsWillBePerformed" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaterialRightsExerciseOrExpiryTerm" xlink:label="bcyc_MaterialRightsExerciseOrExpiryTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_MaterialRightsExerciseOrExpiryTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" xlink:label="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="15" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_RevenueRecognitionAllocatedDeterminedTransactionPrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_EstimatedVariableConsiderationNoLongerConstrained" xlink:label="bcyc_EstimatedVariableConsiderationNoLongerConstrained" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="16" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_EstimatedVariableConsiderationNoLongerConstrained" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" xlink:label="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="17" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborativeArrangementUpfrontCashPaymentReceived" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsSummaryOfContractAssetsAndLiabilitiesDetails" xlink:type="extended" xlink:title="99940911 - Disclosure - Significant agreements - Summary of Contract Assets and Liabilities (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" priority="2" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_BayerMember" xlink:label="bcyc_BayerMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_BayerMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NovartisCollaborationAgreementMember" xlink:label="bcyc_NovartisCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_NovartisCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IonisCollaborationAgreementMember" xlink:label="bcyc_IonisCollaborationAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_IonisCollaborationAgreementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_GenentechMember" xlink:label="bcyc_GenentechMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_GenentechMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IndefiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="us-gaap_IndefiniteLivedIntangibleAssetsByMajorClassAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_IndefiniteLivedIntangibleAssetsByMajorClassAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IndefiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_IndefiniteLivedIntangibleAssetsMajorClassNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_IndefiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="us-gaap_IndefiniteLivedIntangibleAssetsMajorClassNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_DeferredRevenueMaterialRightsOptionMember" xlink:label="bcyc_DeferredRevenueMaterialRightsOptionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IndefiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="bcyc_DeferredRevenueMaterialRightsOptionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ChangeInContractWithCustomerLiabilityAbstract" xlink:label="us-gaap_ChangeInContractWithCustomerLiabilityAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ChangeInContractWithCustomerLiabilityAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerLiability" xlink:label="us-gaap_ContractWithCustomerLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ChangeInContractWithCustomerLiabilityAbstract" xlink:to="us-gaap_ContractWithCustomerLiability" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ContractWithCustomerLiabilityAdditions" xlink:label="bcyc_ContractWithCustomerLiabilityAdditions" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ChangeInContractWithCustomerLiabilityAbstract" xlink:to="bcyc_ContractWithCustomerLiabilityAdditions" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ContractWithCustomerLiabilityDeductions" xlink:label="bcyc_ContractWithCustomerLiabilityDeductions" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ChangeInContractWithCustomerLiabilityAbstract" xlink:to="bcyc_ContractWithCustomerLiabilityDeductions" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ContractWithCustomerLiabilityForeignCurrencyAdjustment" xlink:label="bcyc_ContractWithCustomerLiabilityForeignCurrencyAdjustment" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ChangeInContractWithCustomerLiabilityAbstract" xlink:to="bcyc_ContractWithCustomerLiabilityForeignCurrencyAdjustment" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ChangeInContractWithCustomerAssetAbstract" xlink:label="us-gaap_ChangeInContractWithCustomerAssetAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ChangeInContractWithCustomerAssetAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerAssetNetCurrent" xlink:label="us-gaap_ContractWithCustomerAssetNetCurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ChangeInContractWithCustomerAssetAbstract" xlink:to="us-gaap_ContractWithCustomerAssetNetCurrent" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSignificantAgreementsCancerResearchUkDetails" xlink:type="extended" xlink:title="99940913 - Disclosure - Significant agreements - Cancer Research UK (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable" xlink:label="us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CancerResearchUkBtOneSevenOneEightMember" xlink:label="bcyc_CancerResearchUkBtOneSevenOneEightMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:to="bcyc_CancerResearchUkBtOneSevenOneEightMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersLineItems" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersLineItems" xlink:to="us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ResearchAndDevelopmentArrangementObligationToRepayOtherPartiesAbstract" xlink:label="bcyc_ResearchAndDevelopmentArrangementObligationToRepayOtherPartiesAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersLineItems" xlink:to="bcyc_ResearchAndDevelopmentArrangementObligationToRepayOtherPartiesAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ResearchAndDevelopmentArrangementUpfrontCashPayment" xlink:label="bcyc_ResearchAndDevelopmentArrangementUpfrontCashPayment" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="bcyc_ResearchAndDevelopmentArrangementObligationToRepayOtherPartiesAbstract" xlink:to="bcyc_ResearchAndDevelopmentArrangementUpfrontCashPayment" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_GainLossOnExtinguishmentOfResearchAndDevelopmentFundingLiability" xlink:label="bcyc_GainLossOnExtinguishmentOfResearchAndDevelopmentFundingLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="bcyc_ResearchAndDevelopmentArrangementObligationToRepayOtherPartiesAbstract" xlink:to="bcyc_GainLossOnExtinguishmentOfResearchAndDevelopmentFundingLiability" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureIncomeTaxesReconciliationOfBenefitForIncomeTaxesDetails" xlink:type="extended" xlink:title="99941003 - Disclosure - Income taxes - Reconciliation of benefit for income taxes (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationTable" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxAuthorityAxis" xlink:label="us-gaap_IncomeTaxAuthorityAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_EffectiveIncomeTaxRateReconciliationTable" xlink:to="us-gaap_IncomeTaxAuthorityAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignCountryMember" xlink:label="us-gaap_ForeignCountryMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_ForeignCountryMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_US" xlink:label="country_US" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ForeignCountryMember" xlink:to="country_US" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DomesticCountryMember" xlink:label="us-gaap_DomesticCountryMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_DomesticCountryMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_GB" xlink:label="country_GB" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_DomesticCountryMember" xlink:to="country_GB" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationLineItems" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_EffectiveIncomeTaxRateReconciliationLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxExpenseBenefitContinuingOperationsIncomeTaxReconciliationAbstract" xlink:label="us-gaap_IncomeTaxExpenseBenefitContinuingOperationsIncomeTaxReconciliationAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_EffectiveIncomeTaxRateReconciliationLineItems" xlink:to="us-gaap_IncomeTaxExpenseBenefitContinuingOperationsIncomeTaxReconciliationAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxReconciliationIncomeTaxExpenseBenefitAtFederalStatutoryIncomeTaxRate" xlink:label="us-gaap_IncomeTaxReconciliationIncomeTaxExpenseBenefitAtFederalStatutoryIncomeTaxRate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncomeTaxExpenseBenefitContinuingOperationsIncomeTaxReconciliationAbstract" xlink:to="us-gaap_IncomeTaxReconciliationIncomeTaxExpenseBenefitAtFederalStatutoryIncomeTaxRate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:label="us-gaap_IncomeTaxReconciliationChangeInDeferredTaxAssetsValuationAllowance" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncomeTaxExpenseBenefitContinuingOperationsIncomeTaxReconciliationAbstract" xlink:to="us-gaap_IncomeTaxReconciliationChangeInDeferredTaxAssetsValuationAllowance" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_EffectiveIncomeTaxReconciliationNetLossesSurrenderedForResearchCredit" xlink:label="bcyc_EffectiveIncomeTaxReconciliationNetLossesSurrenderedForResearchCredit" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_IncomeTaxExpenseBenefitContinuingOperationsIncomeTaxReconciliationAbstract" xlink:to="bcyc_EffectiveIncomeTaxReconciliationNetLossesSurrenderedForResearchCredit" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxReconciliationForeignIncomeTaxRateDifferential" xlink:label="us-gaap_IncomeTaxReconciliationForeignIncomeTaxRateDifferential" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_IncomeTaxExpenseBenefitContinuingOperationsIncomeTaxReconciliationAbstract" xlink:to="us-gaap_IncomeTaxReconciliationForeignIncomeTaxRateDifferential" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxReconciliationOtherAdjustments" xlink:label="us-gaap_IncomeTaxReconciliationOtherAdjustments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_IncomeTaxExpenseBenefitContinuingOperationsIncomeTaxReconciliationAbstract" xlink:to="us-gaap_IncomeTaxReconciliationOtherAdjustments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxExpenseBenefit" xlink:label="us-gaap_IncomeTaxExpenseBenefit" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_IncomeTaxExpenseBenefitContinuingOperationsIncomeTaxReconciliationAbstract" xlink:to="us-gaap_IncomeTaxExpenseBenefit" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TaxJurisdictionOfDomicileExtensibleEnumeration" xlink:label="us-gaap_TaxJurisdictionOfDomicileExtensibleEnumeration" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncomeTaxExpenseBenefit" xlink:to="us-gaap_TaxJurisdictionOfDomicileExtensibleEnumeration" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" xlink:label="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_EffectiveIncomeTaxRateReconciliationLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationChangeInDeferredTaxAssetsValuationAllowance" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationChangeInDeferredTaxAssetsValuationAllowance" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationTaxCredits" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationTaxCredits" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationTaxCredits" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_EffectiveIncomeTaxRateReconciliationNetLossesSurrenderedForResearchCredit" xlink:label="bcyc_EffectiveIncomeTaxRateReconciliationNetLossesSurrenderedForResearchCredit" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" xlink:to="bcyc_EffectiveIncomeTaxRateReconciliationNetLossesSurrenderedForResearchCredit" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationChangeInEnactedTaxRate" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationChangeInEnactedTaxRate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationChangeInEnactedTaxRate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationForeignIncomeTaxRateDifferential" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationForeignIncomeTaxRateDifferential" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationForeignIncomeTaxRateDifferential" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationOtherAdjustments" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationOtherAdjustments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationOtherAdjustments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EffectiveIncomeTaxRateContinuingOperations" xlink:label="us-gaap_EffectiveIncomeTaxRateContinuingOperations" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_EffectiveIncomeTaxRateContinuingOperationsTaxRateReconciliationAbstract" xlink:to="us-gaap_EffectiveIncomeTaxRateContinuingOperations" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureIncomeTaxesDetails" xlink:type="extended" xlink:title="99941006 - Disclosure - Income taxes (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLossCarryforwardsTable" xlink:label="us-gaap_OperatingLossCarryforwardsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxAuthorityAxis" xlink:label="us-gaap_IncomeTaxAuthorityAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_OperatingLossCarryforwardsTable" xlink:to="us-gaap_IncomeTaxAuthorityAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StateAndLocalJurisdictionMember" xlink:label="us-gaap_StateAndLocalJurisdictionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_StateAndLocalJurisdictionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLossCarryforwardsLineItems" xlink:label="us-gaap_OperatingLossCarryforwardsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_OperatingLossCarryforwardsLineItems" xlink:to="us-gaap_OperatingLossCarryforwardsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxExpenseBenefit" xlink:label="us-gaap_IncomeTaxExpenseBenefit" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_OperatingLossCarryforwardsLineItems" xlink:to="us-gaap_IncomeTaxExpenseBenefit" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_IncrementalIncomeTaxBenefitOnCompletionOfResearchAndDevelopmentTaxCreditStudy" xlink:label="bcyc_IncrementalIncomeTaxBenefitOnCompletionOfResearchAndDevelopmentTaxCreditStudy" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_OperatingLossCarryforwardsLineItems" xlink:to="bcyc_IncrementalIncomeTaxBenefitOnCompletionOfResearchAndDevelopmentTaxCreditStudy" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncreaseDecreaseInIncomeTaxes" xlink:label="us-gaap_IncreaseDecreaseInIncomeTaxes" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_OperatingLossCarryforwardsLineItems" xlink:to="us-gaap_IncreaseDecreaseInIncomeTaxes" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" xlink:label="us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_OperatingLossCarryforwardsLineItems" xlink:to="us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DeferredTaxAssetsOperatingLossCarryforwardsForeign" xlink:label="us-gaap_DeferredTaxAssetsOperatingLossCarryforwardsForeign" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_OperatingLossCarryforwardsLineItems" xlink:to="us-gaap_DeferredTaxAssetsOperatingLossCarryforwardsForeign" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnrecognizedTaxBenefits" xlink:label="us-gaap_UnrecognizedTaxBenefits" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_OperatingLossCarryforwardsLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefits" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnrecognizedTaxBenefitsIncomeTaxPenaltiesAndInterestAccrued" xlink:label="us-gaap_UnrecognizedTaxBenefitsIncomeTaxPenaltiesAndInterestAccrued" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_OperatingLossCarryforwardsLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefitsIncomeTaxPenaltiesAndInterestAccrued" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NumberOfJurisdictions" xlink:label="bcyc_NumberOfJurisdictions" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_OperatingLossCarryforwardsLineItems" xlink:to="bcyc_NumberOfJurisdictions" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureCommitmentsAndContingenciesOperatingLeasesAndFinanceLeasesDetails" xlink:type="extended" xlink:title="99941101 - Disclosure - Commitments and contingencies - Operating Leases and Finance Leases (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeLeaseDescriptionTable" xlink:label="us-gaap_LesseeLeaseDescriptionTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_LesseeLeaseDescriptionTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_OfficeAndLaboratorySpaceInCambridgeMassachusettsMember" xlink:label="bcyc_OfficeAndLaboratorySpaceInCambridgeMassachusettsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="bcyc_OfficeAndLaboratorySpaceInCambridgeMassachusettsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NewOfficeAndLaboratorySpaceInCambridgeUnitedKingdomMember" xlink:label="bcyc_NewOfficeAndLaboratorySpaceInCambridgeUnitedKingdomMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="bcyc_NewOfficeAndLaboratorySpaceInCambridgeUnitedKingdomMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_OfficeAndLaboratorySpaceInBuilding900BabrahamResearchCampusCambridgeMember" xlink:label="bcyc_OfficeAndLaboratorySpaceInBuilding900BabrahamResearchCampusCambridgeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="bcyc_OfficeAndLaboratorySpaceInBuilding900BabrahamResearchCampusCambridgeMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeLeaseDescriptionLineItems" xlink:label="us-gaap_LesseeLeaseDescriptionLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LesseeLeaseDescriptionTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_LesseeOperatingLeaseContractualTerm" xlink:label="bcyc_LesseeOperatingLeaseContractualTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="bcyc_LesseeOperatingLeaseContractualTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseTermOfContract" xlink:label="us-gaap_LesseeOperatingLeaseTermOfContract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LesseeOperatingLeaseTermOfContract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_LeaseRentFreePeriod" xlink:label="bcyc_LeaseRentFreePeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="bcyc_LeaseRentFreePeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseRenewalTerm" xlink:label="us-gaap_LesseeOperatingLeaseRenewalTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LesseeOperatingLeaseRenewalTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RightOfUseAssetObtainedInExchangeForOperatingLeaseLiability" xlink:label="us-gaap_RightOfUseAssetObtainedInExchangeForOperatingLeaseLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_RightOfUseAssetObtainedInExchangeForOperatingLeaseLiability" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GainLossOnTerminationOfLease" xlink:label="us-gaap_GainLossOnTerminationOfLease" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_GainLossOnTerminationOfLease" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseRightOfUseAsset" xlink:label="us-gaap_FinanceLeaseRightOfUseAsset" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_FinanceLeaseRightOfUseAsset" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseRightOfUseAssetStatementOfFinancialPositionExtensibleList" xlink:label="us-gaap_FinanceLeaseRightOfUseAssetStatementOfFinancialPositionExtensibleList" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FinanceLeaseRightOfUseAsset" xlink:to="us-gaap_FinanceLeaseRightOfUseAssetStatementOfFinancialPositionExtensibleList" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiability" xlink:label="us-gaap_FinanceLeaseLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_FinanceLeaseLiability" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiabilityStatementOfFinancialPositionExtensibleList" xlink:label="us-gaap_FinanceLeaseLiabilityStatementOfFinancialPositionExtensibleList" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FinanceLeaseLiability" xlink:to="us-gaap_FinanceLeaseLiabilityStatementOfFinancialPositionExtensibleList" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureCommitmentsAndContingenciesLeasesMaturitiesOfOperatingLeasesDetails" xlink:type="extended" xlink:title="99941103 - Disclosure - Commitments and contingencies - Leases, Maturities of Operating Leases (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_AgreementWithThirdPartyMember" xlink:label="bcyc_AgreementWithThirdPartyMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="bcyc_AgreementWithThirdPartyMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:label="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueNextTwelveMonths" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueNextTwelveMonths" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFive" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFive" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFive" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueAfterYearFive" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueAfterYearFive" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueAfterYearFive" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiability" xlink:label="us-gaap_OperatingLeaseLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_OperatingLeaseLiability" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiabilityCurrent" xlink:label="us-gaap_OperatingLeaseLiabilityCurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_OperatingLeaseLiabilityCurrent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:label="us-gaap_OperatingLeaseLiabilityNoncurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_OperatingLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_OperatingLeaseLiabilityNoncurrent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiabilityCurrentStatementOfFinancialPositionExtensibleList" xlink:label="us-gaap_OperatingLeaseLiabilityCurrentStatementOfFinancialPositionExtensibleList" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:to="us-gaap_OperatingLeaseLiabilityCurrentStatementOfFinancialPositionExtensibleList" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" xlink:label="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDueNextTwelveMonths" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDueNextTwelveMonths" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDueYearTwo" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDueYearTwo" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDueYearTwo" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDueYearThree" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDueYearThree" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDueYearThree" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDueYearFour" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDueYearFour" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDueYearFour" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiabilityUndiscountedExcessAmount" xlink:label="us-gaap_FinanceLeaseLiabilityUndiscountedExcessAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_FinanceLeaseLiabilityUndiscountedExcessAmount" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiability" xlink:label="us-gaap_FinanceLeaseLiability" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_FinanceLeaseLiability" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiabilityCurrent" xlink:label="us-gaap_FinanceLeaseLiabilityCurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_FinanceLeaseLiabilityCurrent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinanceLeaseLiabilityNoncurrent" xlink:label="us-gaap_FinanceLeaseLiabilityNoncurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_FinanceLeaseLiabilitiesPaymentsDueAbstract" xlink:to="us-gaap_FinanceLeaseLiabilityNoncurrent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_MaximumDaysAllowedForCancellationOfContractsPriorWrittenNotice" xlink:label="bcyc_MaximumDaysAllowedForCancellationOfContractsPriorWrittenNotice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_MaximumDaysAllowedForCancellationOfContractsPriorWrittenNotice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_CollaborationAgreementFutureMilestoneContingentPaymentsPayable" xlink:label="bcyc_CollaborationAgreementFutureMilestoneContingentPaymentsPayable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="bcyc_CollaborationAgreementFutureMilestoneContingentPaymentsPayable" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureNetLossPerShareSecuritiesExcludedFromDilutedPerShareCalculationDetails" xlink:type="extended" xlink:title="99941202 - Disclosure - Net loss per share - Securities excluded from the diluted per share calculation (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockMember" xlink:label="us-gaap_RestrictedStockMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_RestrictedStockMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureBenefitPlansDetails" xlink:type="extended" xlink:title="99941301 - Disclosure - Benefit plans (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DefinedContributionPlanTable" xlink:label="us-gaap_DefinedContributionPlanTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetirementPlanSponsorLocationAxis" xlink:label="us-gaap_RetirementPlanSponsorLocationAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_DefinedContributionPlanTable" xlink:to="us-gaap_RetirementPlanSponsorLocationAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetirementPlanSponsorLocationDomain" xlink:label="us-gaap_RetirementPlanSponsorLocationDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RetirementPlanSponsorLocationAxis" xlink:to="us-gaap_RetirementPlanSponsorLocationDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DomesticPlanMember" xlink:label="us-gaap_DomesticPlanMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RetirementPlanSponsorLocationDomain" xlink:to="us-gaap_DomesticPlanMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_US" xlink:label="country_US" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_DomesticPlanMember" xlink:to="country_US" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignPlanMember" xlink:label="us-gaap_ForeignPlanMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RetirementPlanSponsorLocationDomain" xlink:to="us-gaap_ForeignPlanMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetirementPlanNameAxis" xlink:label="us-gaap_RetirementPlanNameAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_DefinedContributionPlanTable" xlink:to="us-gaap_RetirementPlanNameAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetirementPlanNameDomain" xlink:label="us-gaap_RetirementPlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RetirementPlanNameAxis" xlink:to="us-gaap_RetirementPlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_Plan401KMember" xlink:label="bcyc_Plan401KMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RetirementPlanNameDomain" xlink:to="bcyc_Plan401KMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_UKPlanMember" xlink:label="bcyc_UKPlanMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RetirementPlanNameDomain" xlink:to="bcyc_UKPlanMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DefinedContributionPlanDisclosureLineItems" xlink:label="us-gaap_DefinedContributionPlanDisclosureLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_DefinedContributionPlanDisclosureLineItems" xlink:to="us-gaap_DefinedContributionPlanTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DefinedContributionPlanCostRecognized" xlink:label="us-gaap_DefinedContributionPlanCostRecognized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_DefinedContributionPlanDisclosureLineItems" xlink:to="us-gaap_DefinedContributionPlanCostRecognized" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureRelatedPartyTransactionsDetails" xlink:type="extended" xlink:title="99941401 - Disclosure - Related party transactions (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:label="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_AffiliatedEntityMember" xlink:label="srt_AffiliatedEntityMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="srt_AffiliatedEntityMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:to="srt_CounterpartyNameAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ConsultancyServicesWithStoneSunnyIslesInc.Member" xlink:label="bcyc_ConsultancyServicesWithStoneSunnyIslesInc.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="bcyc_ConsultancyServicesWithStoneSunnyIslesInc.Member" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_BakerBrothersAdvisorsLpMember" xlink:label="bcyc_BakerBrothersAdvisorsLpMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="bcyc_BakerBrothersAdvisorsLpMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:to="us-gaap_StatementClassOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_NonVotingOrdinarySharesMember" xlink:label="bcyc_NonVotingOrdinarySharesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="bcyc_NonVotingOrdinarySharesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RelatedPartyTransactionLineItems" xlink:label="us-gaap_RelatedPartyTransactionLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="us-gaap_GeneralAndAdministrativeExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="us-gaap_CommonStockSharesIssued" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSegmentsAndGeographicInformationDetails" xlink:type="extended" xlink:title="99941501 - Disclosure - Segments and geographic information (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" xlink:label="us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis" xlink:label="srt_StatementGeographicalAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" xlink:to="srt_StatementGeographicalAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_US" xlink:label="country_US" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_SegmentGeographicalDomain" xlink:to="country_US" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_GB" xlink:label="country_GB" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_SegmentGeographicalDomain" xlink:to="country_GB" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:label="us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:to="us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NumberOfOperatingSegments" xlink:label="us-gaap_NumberOfOperatingSegments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:to="us-gaap_NumberOfOperatingSegments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NumberOfReportableSegments" xlink:label="us-gaap_NumberOfReportableSegments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:to="us-gaap_NumberOfReportableSegments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NoncurrentAssets" xlink:label="us-gaap_NoncurrentAssets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:to="us-gaap_NoncurrentAssets" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSegmentsAndGeographicInformationSignificantSegmentExpensesDetails" xlink:type="extended" xlink:title="99941502 - Disclosure - Segments and geographic information - Significant segment expenses (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:label="us-gaap_StatementBusinessSegmentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_StatementBusinessSegmentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_SingleReportableSegmentMember" xlink:label="bcyc_SingleReportableSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentDomain" xlink:to="bcyc_SingleReportableSegmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedCostsOfUnprovedPropertiesExcludedFromAmortizationByPropertyOrProjectAxis" xlink:label="us-gaap_CapitalizedCostsOfUnprovedPropertiesExcludedFromAmortizationByPropertyOrProjectAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_CapitalizedCostsOfUnprovedPropertiesExcludedFromAmortizationByPropertyOrProjectAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProjectMember" xlink:label="us-gaap_ProjectMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_CapitalizedCostsOfUnprovedPropertiesExcludedFromAmortizationByPropertyOrProjectAxis" xlink:to="us-gaap_ProjectMember" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_InternalProgramProductCandidateBicycleToxinConjugateMoleculeBT8009Member" xlink:label="bcyc_InternalProgramProductCandidateBicycleToxinConjugateMoleculeBT8009Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ProjectMember" xlink:to="bcyc_InternalProgramProductCandidateBicycleToxinConjugateMoleculeBT8009Member" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_InternalProgramProductCandidateBicycleToxinConjugateMoleculeBT5528Member" xlink:label="bcyc_InternalProgramProductCandidateBicycleToxinConjugateMoleculeBT5528Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ProjectMember" xlink:to="bcyc_InternalProgramProductCandidateBicycleToxinConjugateMoleculeBT5528Member" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_InternalProgramBicycleTumorTargetedImmuneCellAgonistsBT7480Member" xlink:label="bcyc_InternalProgramBicycleTumorTargetedImmuneCellAgonistsBT7480Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ProjectMember" xlink:to="bcyc_InternalProgramBicycleTumorTargetedImmuneCellAgonistsBT7480Member" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentReportingInformationLineItems" xlink:label="us-gaap_SegmentReportingInformationLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingExpensesAbstract" xlink:label="us-gaap_OperatingExpensesAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_OperatingExpensesAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:label="us-gaap_ResearchAndDevelopmentExpenseAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_ResearchAndDevelopmentExpenseAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ProjectSpecificCosts" xlink:label="bcyc_ProjectSpecificCosts" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_ProjectSpecificCosts" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ResearchAndDevelopmentDiscoveryPlatformAndOtherExpenses" xlink:label="bcyc_ResearchAndDevelopmentDiscoveryPlatformAndOtherExpenses" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_ResearchAndDevelopmentDiscoveryPlatformAndOtherExpenses" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ResearchAndDevelopmentEmployeeAndContractorRelatedExpenses" xlink:label="bcyc_ResearchAndDevelopmentEmployeeAndContractorRelatedExpenses" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_ResearchAndDevelopmentEmployeeAndContractorRelatedExpenses" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ResearchAndDevelopmentShareBasedPaymentArrangementExpense" xlink:label="bcyc_ResearchAndDevelopmentShareBasedPaymentArrangementExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_ResearchAndDevelopmentShareBasedPaymentArrangementExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_FacilityExpenses" xlink:label="bcyc_FacilityExpenses" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_FacilityExpenses" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_ResearchAndDevelopmentTaxCreditsIncentivesAndGovernmentGrants" xlink:label="bcyc_ResearchAndDevelopmentTaxCreditsIncentivesAndGovernmentGrants" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="bcyc_ResearchAndDevelopmentTaxCreditsIncentivesAndGovernmentGrants" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ResearchAndDevelopmentExpenseAbstract" xlink:to="us-gaap_ResearchAndDevelopmentExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpenseAbstract" xlink:label="us-gaap_GeneralAndAdministrativeExpenseAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_GeneralAndAdministrativeExpenseAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LaborAndRelatedExpense" xlink:label="us-gaap_LaborAndRelatedExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_GeneralAndAdministrativeExpenseAbstract" xlink:to="us-gaap_LaborAndRelatedExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProfessionalFees" xlink:label="us-gaap_ProfessionalFees" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_GeneralAndAdministrativeExpenseAbstract" xlink:to="us-gaap_ProfessionalFees" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherGeneralAndAdministrativeExpense" xlink:label="us-gaap_OtherGeneralAndAdministrativeExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_GeneralAndAdministrativeExpenseAbstract" xlink:to="us-gaap_OtherGeneralAndAdministrativeExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_GeneralAndAdministrativeShareBasedPaymentArrangementExpense" xlink:label="bcyc_GeneralAndAdministrativeShareBasedPaymentArrangementExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_GeneralAndAdministrativeExpenseAbstract" xlink:to="bcyc_GeneralAndAdministrativeShareBasedPaymentArrangementExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignCurrencyTransactionGainLossBeforeTax" xlink:label="us-gaap_ForeignCurrencyTransactionGainLossBeforeTax" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_GeneralAndAdministrativeExpenseAbstract" xlink:to="us-gaap_ForeignCurrencyTransactionGainLossBeforeTax" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_GeneralAndAdministrativeExpenseAbstract" xlink:to="us-gaap_GeneralAndAdministrativeExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingExpenses" xlink:label="us-gaap_OperatingExpenses" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_OperatingExpenses" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentReportingOtherItemAmount" xlink:label="us-gaap_SegmentReportingOtherItemAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_SegmentReportingOtherItemAmount" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_NetIncomeLoss" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentReportingOtherItemCompositionDescription" xlink:label="us-gaap_SegmentReportingOtherItemCompositionDescription" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_SegmentReportingOtherItemCompositionDescription" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.bicycletherapeutics.com/role/DisclosureSubsequentEventsDetails" xlink:type="extended" xlink:title="99941601 - Disclosure - Subsequent events (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTable" xlink:label="us-gaap_SubsequentEventTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_SubsequentEventTable" xlink:to="us-gaap_SubsequentEventTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventLineItems" xlink:label="us-gaap_SubsequentEventLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_SubsequentEventTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringAndRelatedCostNumberOfPositionsEliminatedPeriodPercent" xlink:label="us-gaap_RestructuringAndRelatedCostNumberOfPositionsEliminatedPeriodPercent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_RestructuringAndRelatedCostNumberOfPositionsEliminatedPeriodPercent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="bcyc-20251231.xsd#bcyc_RestructuringAndRelatedCostExpectedCostReductionPercent" xlink:label="bcyc_RestructuringAndRelatedCostExpectedCostReductionPercent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="bcyc_RestructuringAndRelatedCostExpectedCostReductionPercent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestructuringAndRelatedCostExpectedCost1" xlink:label="us-gaap_RestructuringAndRelatedCostExpectedCost1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_RestructuringAndRelatedCostExpectedCost1" priority="1" use="optional" />
  </definitionLink>
</link:linkbase>
