<?xml version="1.0" encoding="us-ascii"?>
<!-- XBRL document created with Praxa -->
<!-- Copyright (c) 2026 Praxa LLC. All Rights Reserved.-->
<!-- Creation Date: 2026-11-13T20:39:36.5357+00:00 -->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" xlink:type="simple" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" xlink:type="simple" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" xlink:type="simple" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" xlink:type="simple" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" xlink:type="simple" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/notAll" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#notAll" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/OrganizationDescriptionofBusinessGoingConcernandSignificantRisksandUncertaintiesDetails" xlink:href="pfsa-20260930.xsd#OrganizationDescriptionofBusinessGoingConcernandSignificantRisksandUncertaintiesDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/FairValueMeasurementScheduleofFairValueoftheCompanysLevel3RelatedPartyConvertibleNotesPayableDetails" xlink:href="pfsa-20260930.xsd#FairValueMeasurementScheduleofFairValueoftheCompanysLevel3RelatedPartyConvertibleNotesPayableDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/SummaryofSignificantAccountingPoliciesDetails" xlink:href="pfsa-20260930.xsd#SummaryofSignificantAccountingPoliciesDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/DebtDetails" xlink:href="pfsa-20260930.xsd#DebtDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/FairValueMeasurementScheduleofInputsintotheMonteCarloSimulationModelfortheWarrantLiabilitiesandConvertibleLoansPayableDetails" xlink:href="pfsa-20260930.xsd#FairValueMeasurementScheduleofInputsintotheMonteCarloSimulationModelfortheWarrantLiabilitiesandConvertibleLoansPayableDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/FairValueMeasurementScheduleofFinancialInstrumentsthatareMeasuredatFairValueonaRecurringBasisDetails" xlink:href="pfsa-20260930.xsd#FairValueMeasurementScheduleofFinancialInstrumentsthatareMeasuredatFairValueonaRecurringBasisDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/FairValueMeasurementScheduleofFinancialAssetsandLiabilitiesMeasuredatFairValueonaRecurringBasisDetails" xlink:href="pfsa-20260930.xsd#FairValueMeasurementScheduleofFinancialAssetsandLiabilitiesMeasuredatFairValueonaRecurringBasisDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/NetLossPerShareAttributabletoCommonStockholdersScheduleofDilutedNetLossPerShareAttributabletoCommonStockholdersDetails" xlink:href="pfsa-20260930.xsd#NetLossPerShareAttributabletoCommonStockholdersScheduleofDilutedNetLossPerShareAttributabletoCommonStockholdersDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/DebtScheduleofDebtInstrumentsandtheirChangesDetails" xlink:href="pfsa-20260930.xsd#DebtScheduleofDebtInstrumentsandtheirChangesDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/RelatedPartyTransactionsDetails" xlink:href="pfsa-20260930.xsd#RelatedPartyTransactionsDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/StockholdersEquityDeficitScheduleofReservedSharesofCommonStockforFutureIssuanceDetails" xlink:href="pfsa-20260930.xsd#StockholdersEquityDeficitScheduleofReservedSharesofCommonStockforFutureIssuanceDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/StockholdersEquityDeficitDetails" xlink:href="pfsa-20260930.xsd#StockholdersEquityDeficitDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/CommitmentsandContingenciesDetails" xlink:href="pfsa-20260930.xsd#CommitmentsandContingenciesDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/ProposedAcquisitionofG3Details" xlink:href="pfsa-20260930.xsd#ProposedAcquisitionofG3Details" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/SubsequentEventsDetails" xlink:href="pfsa-20260930.xsd#SubsequentEventsDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/CommonStockWarrantsDetails" xlink:href="pfsa-20260930.xsd#CommonStockWarrantsDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentDetails" xlink:href="pfsa-20260930.xsd#SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/NetLossPerShareAttributabletoCommonStockholdersDetails" xlink:href="pfsa-20260930.xsd#NetLossPerShareAttributabletoCommonStockholdersDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/RelatedPartyTransactionsScheduleofRelatedPartyTransactionsDetails" xlink:href="pfsa-20260930.xsd#RelatedPartyTransactionsScheduleofRelatedPartyTransactionsDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentParentheticalsDetails" xlink:href="pfsa-20260930.xsd#SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentParentheticalsDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/StockholdersEquityDeficitScheduleofConvertiblePreferredStockDetails" xlink:href="pfsa-20260930.xsd#StockholdersEquityDeficitScheduleofConvertiblePreferredStockDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/CondensedConsolidatedStatementsofConvertiblePreferredStockandStockholdersEquityDeficitUnaudited" xlink:href="pfsa-20260930.xsd#CondensedConsolidatedStatementsofConvertiblePreferredStockandStockholdersEquityDeficitUnaudited" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/FairValueMeasurementScheduleofKeyInputsintotheMonteCarloSimulationModelfortheConvertiblePromissoryNoteDetails" xlink:href="pfsa-20260930.xsd#FairValueMeasurementScheduleofKeyInputsintotheMonteCarloSimulationModelfortheConvertiblePromissoryNoteDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/CondensedConsolidatedBalanceSheetsParentheticals" xlink:href="pfsa-20260930.xsd#CondensedConsolidatedBalanceSheetsParentheticals" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/FairValueMeasurementScheduleofChangesintheFairValueoftheCompanysLevel3LoansPayableDetails" xlink:href="pfsa-20260930.xsd#FairValueMeasurementScheduleofChangesintheFairValueoftheCompanysLevel3LoansPayableDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/FairValueMeasurementDetails" xlink:href="pfsa-20260930.xsd#FairValueMeasurementDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/CommonStockWarrantsScheduleofClassesofWarrantsDetails" xlink:href="pfsa-20260930.xsd#CommonStockWarrantsScheduleofClassesofWarrantsDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/StockOptionPlanDetails" xlink:href="pfsa-20260930.xsd#StockOptionPlanDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/CommonStockWarrantsScheduleofFollowingKeyInputsasAffectedbytheReverseStockSplitsDetails" xlink:href="pfsa-20260930.xsd#CommonStockWarrantsScheduleofFollowingKeyInputsasAffectedbytheReverseStockSplitsDetails" xlink:type="simple" />
  <link:roleRef roleURI="http://profusa.com/20260930/role/CondensedConsolidatedBalanceSheets" xlink:href="pfsa-20260930.xsd#CondensedConsolidatedBalanceSheets" xlink:type="simple" />
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/OrganizationDescriptionofBusinessGoingConcernandSignificantRisksandUncertaintiesDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PIPEConvertibleNoteMember" xlink:label="pfsa_PIPEConvertibleNoteMember_1465" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2017" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ELOCPurchaseAgreementMember" xlink:label="pfsa_ELOCPurchaseAgreementMember_2018" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember_1462" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AscentConvertibleNoteAgreementMember" xlink:label="pfsa_AscentConvertibleNoteAgreementMember_2213" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OrganizationConsolidationAndPresentationOfFinancialStatementsAbstract" xlink:label="us-gaap_OrganizationConsolidationAndPresentationOfFinancialStatementsAbstract_1458" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityIncorporationDateOfIncorporation" xlink:label="dei_EntityIncorporationDateOfIncorporation_1470" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Cash" xlink:label="us-gaap_Cash_1480" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction" xlink:label="us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_2127" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueIssuedForServices" xlink:label="us-gaap_StockIssuedDuringPeriodValueIssuedForServices_1479" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_OrganizationDescriptionofBusinessGoingConcernandSignificantRisksandUncertaintiesDetailsTable" xlink:label="pfsa_OrganizationDescriptionofBusinessGoingConcernandSignificantRisksandUncertaintiesDetailsTable_1459" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1461" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis_1463" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1464" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_WorkingCapitalDeficit" xlink:label="pfsa_WorkingCapitalDeficit_1471" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss_1472" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleNotesPayable" xlink:label="us-gaap_ConvertibleNotesPayable_2212" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivities_1473" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="us-gaap_DebtInstrumentFaceAmount_1474" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis_2016" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_1460" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockConsiderationReceivedOnTransaction" xlink:label="us-gaap_SaleOfStockConsiderationReceivedOnTransaction_1476" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2017_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1461_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1464_default" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1464" xlink:to="pfsa_PIPEConvertibleNoteMember_1465" />
    <link:definitionArc xlink:type="arc" order="112.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis_2016" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2017" />
    <link:definitionArc xlink:type="arc" order="113.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2017" xlink:to="pfsa_ELOCPurchaseAgreementMember_2018" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1461" xlink:to="us-gaap_CommonStockMember_1462" />
    <link:definitionArc xlink:type="arc" order="277.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2017" xlink:to="pfsa_AscentConvertibleNoteAgreementMember_2213" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="dei_EntityIncorporationDateOfIncorporation_1470" />
    <link:definitionArc xlink:type="arc" order="22.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="us-gaap_Cash_1480" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_2127" />
    <link:definitionArc xlink:type="arc" order="21.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="us-gaap_StockIssuedDuringPeriodValueIssuedForServices_1479" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="pfsa_OrganizationDescriptionofBusinessGoingConcernandSignificantRisksandUncertaintiesDetailsTable_1459" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_1460" xlink:to="us-gaap_ClassOfStockDomain_1461" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_OrganizationDescriptionofBusinessGoingConcernandSignificantRisksandUncertaintiesDetailsTable_1459" xlink:to="us-gaap_DebtInstrumentAxis_1463" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis_1463" xlink:to="us-gaap_DebtInstrumentNameDomain_1464" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="pfsa_WorkingCapitalDeficit_1471" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="us-gaap_NetIncomeLoss_1472" />
    <link:definitionArc xlink:type="arc" order="243.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="us-gaap_ConvertibleNotesPayable_2212" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivities_1473" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="us-gaap_DebtInstrumentFaceAmount_1474" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_OrganizationDescriptionofBusinessGoingConcernandSignificantRisksandUncertaintiesDetailsTable_1459" xlink:to="us-gaap_TypeOfArrangementAxis_2016" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_OrganizationDescriptionofBusinessGoingConcernandSignificantRisksandUncertaintiesDetailsTable_1459" xlink:to="us-gaap_StatementClassOfStockAxis_1460" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_1469" xlink:to="us-gaap_SaleOfStockConsiderationReceivedOnTransaction_1476" />
    <link:definitionArc xlink:type="arc" order="112.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis_2016" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2017_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_1460" xlink:to="us-gaap_ClassOfStockDomain_1461_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis_1463" xlink:to="us-gaap_DebtInstrumentNameDomain_1464_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/FairValueMeasurementScheduleofFairValueoftheCompanysLevel3RelatedPartyConvertibleNotesPayableDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TaslyConvertibleNoteRelatedPartyMember" xlink:label="pfsa_TaslyConvertibleNoteRelatedPartyMember_1269" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_RelatedPartyConvertiblePromissoryNoteMember" xlink:label="pfsa_RelatedPartyConvertiblePromissoryNoteMember_1270" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TotalLevel3RelatedPartyConvertibleNotesMember" xlink:label="pfsa_TotalLevel3RelatedPartyConvertibleNotesMember_1271" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTable" xlink:label="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTable_1267" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis_1268" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems" xlink:label="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1272" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue" xlink:label="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue_1273" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings" xlink:label="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings_1274" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConversionOfDebtToEquity" xlink:label="pfsa_ConversionOfDebtToEquity_2244" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DebtExtinguishment" xlink:label="pfsa_DebtExtinguishment_2245" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AssumptionOfRelatedPartyConvertibleNoteUponClosingOfTheBusinessCombination" xlink:label="pfsa_AssumptionOfRelatedPartyConvertibleNoteUponClosingOfTheBusinessCombination_2246" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_GainOnChangeInTheFairValueOfRelatedPartyConvertibleNotes" xlink:label="pfsa_GainOnChangeInTheFairValueOfRelatedPartyConvertibleNotes_2247" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1999" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfFairValueOfTheCompanySLevel3RelatedPartyConvertibleDebtPayableAbstract" xlink:label="pfsa_ScheduleOfFairValueOfTheCompanySLevel3RelatedPartyConvertibleDebtPayableAbstract_1266" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1999_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1999" xlink:to="pfsa_TaslyConvertibleNoteRelatedPartyMember_1269" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1999" xlink:to="pfsa_RelatedPartyConvertiblePromissoryNoteMember_1270" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1999" xlink:to="pfsa_TotalLevel3RelatedPartyConvertibleNotesMember_1271" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1272" xlink:to="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTable_1267" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTable_1267" xlink:to="us-gaap_FinancialInstrumentAxis_1268" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1272" xlink:to="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue_1273" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1272" xlink:to="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings_1274" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1272" xlink:to="pfsa_ConversionOfDebtToEquity_2244" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1272" xlink:to="pfsa_DebtExtinguishment_2245" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1272" xlink:to="pfsa_AssumptionOfRelatedPartyConvertibleNoteUponClosingOfTheBusinessCombination_2246" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1272" xlink:to="pfsa_GainOnChangeInTheFairValueOfRelatedPartyConvertibleNotes_2247" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FinancialInstrumentAxis_1268" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1999" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FinancialInstrumentAxis_1268" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1999_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/SummaryofSignificantAccountingPoliciesDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AscentPIPENotesMember" xlink:label="pfsa_AscentPIPENotesMember_2460" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ScenarioPreviouslyReportedMember" xlink:label="srt_ScenarioPreviouslyReportedMember_2470" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2129" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ELOCPurchaseAgreementMember" xlink:label="pfsa_ELOCPurchaseAgreementMember_2130" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FinancingArrangementMember" xlink:label="pfsa_FinancingArrangementMember_2499" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PIPEAgreementMember" xlink:label="pfsa_PIPEAgreementMember_2200" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_JulyReverseStockSplitMember" xlink:label="pfsa_JulyReverseStockSplitMember_2112" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember_1487" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AugustReverseStockSplitMember" xlink:label="pfsa_AugustReverseStockSplitMember_2286" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FebruaryReverseStockSplitMember" xlink:label="pfsa_FebruaryReverseStockSplitMember_2287" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ELOCWarrantsMember" xlink:label="pfsa_ELOCWarrantsMember_1490" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentIssuedPrincipal" xlink:label="us-gaap_DebtInstrumentIssuedPrincipal_2462" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtPercentageBearingFixedInterestRate" xlink:label="us-gaap_ShortTermDebtPercentageBearingFixedInterestRate_2463" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis_2464" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_2466" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss_2467" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RestatementAxis" xlink:label="srt_RestatementAxis_2468" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RestatementDomain" xlink:label="srt_RestatementDomain_2469" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentMaturityDate" xlink:label="us-gaap_DebtInstrumentMaturityDate_2471" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_2472" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis_2128" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TrueupCommonSharesIssued" xlink:label="pfsa_TrueupCommonSharesIssued_2131" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountingPoliciesAbstract" xlink:label="us-gaap_AccountingPoliciesAbstract_1483" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SummaryofSignificantAccountingPoliciesDetailsTable" xlink:label="pfsa_SummaryofSignificantAccountingPoliciesDetailsTable_1484" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_1485" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare_2169" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeFairValueOfDerivativeNet" xlink:label="us-gaap_DerivativeFairValueOfDerivativeNet_2198" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="us-gaap_DebtInstrumentFaceAmount_2199" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain_2111" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TaxesOther" xlink:label="us-gaap_TaxesOther_2024" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TaxesPayableCurrentAndNoncurrent" xlink:label="us-gaap_TaxesPayableCurrentAndNoncurrent_2025" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapitalCommonStock" xlink:label="us-gaap_AdditionalPaidInCapitalCommonStock_2026" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherLiabilities" xlink:label="us-gaap_OtherLiabilities_2027" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PercentageOfPurchasePrice" xlink:label="pfsa_PercentageOfPurchasePrice_2029" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PercentageOfAdditionalShares" xlink:label="pfsa_PercentageOfAdditionalShares_2030" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1486" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis_1488" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit_2275" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued_2031" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AdvanceNoticeShares" xlink:label="pfsa_AdvanceNoticeShares_2032" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_GrossProceeds" xlink:label="pfsa_GrossProceeds_2034" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ShareIssuanceLiability" xlink:label="pfsa_ShareIssuanceLiability_2035" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InvestmentOwnedBalancePrincipalAmount" xlink:label="us-gaap_InvestmentOwnedBalancePrincipalAmount_2036" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PaymentsOfFinancingCosts" xlink:label="us-gaap_PaymentsOfFinancingCosts_2038" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsFairValueAdjustment" xlink:label="us-gaap_AssetsFairValueAdjustment_2039" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_1489" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ChangeInAccountingEstimateLineItems" xlink:label="us-gaap_ChangeInAccountingEstimateLineItems_1491" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityReverseStockSplit" xlink:label="us-gaap_StockholdersEquityReverseStockSplit_1492" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionAxis" xlink:label="us-gaap_RelatedPartyTransactionAxis_2110" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesIssuedPricePerShare" xlink:label="us-gaap_SharesIssuedPricePerShare_2023" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2129_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_2466_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RestatementDomain" xlink:label="srt_RestatementDomain_2469_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain_2111_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1486_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_1489_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_2466" xlink:to="pfsa_AscentPIPENotesMember_2460" />
    <link:definitionArc xlink:type="arc" order="462.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RestatementDomain_2469" xlink:to="srt_ScenarioPreviouslyReportedMember_2470" />
    <link:definitionArc xlink:type="arc" order="211.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis_2128" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2129" />
    <link:definitionArc xlink:type="arc" order="212.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2129" xlink:to="pfsa_ELOCPurchaseAgreementMember_2130" />
    <link:definitionArc xlink:type="arc" order="486.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_2466" xlink:to="pfsa_FinancingArrangementMember_2499" />
    <link:definitionArc xlink:type="arc" order="267.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2129" xlink:to="pfsa_PIPEAgreementMember_2200" />
    <link:definitionArc xlink:type="arc" order="197.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain_2111" xlink:to="pfsa_JulyReverseStockSplitMember_2112" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1486" xlink:to="us-gaap_CommonStockMember_1487" />
    <link:definitionArc xlink:type="arc" order="322.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain_2111" xlink:to="pfsa_AugustReverseStockSplitMember_2286" />
    <link:definitionArc xlink:type="arc" order="323.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain_2111" xlink:to="pfsa_FebruaryReverseStockSplitMember_2287" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1489" xlink:to="pfsa_ELOCWarrantsMember_1490" />
    <link:definitionArc xlink:type="arc" order="455.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_DebtInstrumentIssuedPrincipal_2462" />
    <link:definitionArc xlink:type="arc" order="456.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_ShortTermDebtPercentageBearingFixedInterestRate_2463" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SummaryofSignificantAccountingPoliciesDetailsTable_1484" xlink:to="us-gaap_DebtInstrumentAxis_2464" />
    <link:definitionArc xlink:type="arc" order="459.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis_2464" xlink:to="us-gaap_DebtInstrumentNameDomain_2466" />
    <link:definitionArc xlink:type="arc" order="457.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_NetIncomeLoss_2467" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SummaryofSignificantAccountingPoliciesDetailsTable_1484" xlink:to="srt_RestatementAxis_2468" />
    <link:definitionArc xlink:type="arc" order="461.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RestatementAxis_2468" xlink:to="srt_RestatementDomain_2469" />
    <link:definitionArc xlink:type="arc" order="463.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_DebtInstrumentMaturityDate_2471" />
    <link:definitionArc xlink:type="arc" order="464.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_2472" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SummaryofSignificantAccountingPoliciesDetailsTable_1484" xlink:to="us-gaap_TypeOfArrangementAxis_2128" />
    <link:definitionArc xlink:type="arc" order="25.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="pfsa_TrueupCommonSharesIssued_2131" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="pfsa_SummaryofSignificantAccountingPoliciesDetailsTable_1484" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SummaryofSignificantAccountingPoliciesDetailsTable_1484" xlink:to="us-gaap_StatementClassOfStockAxis_1485" />
    <link:definitionArc xlink:type="arc" order="243.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare_2169" />
    <link:definitionArc xlink:type="arc" order="265.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_DerivativeFairValueOfDerivativeNet_2198" />
    <link:definitionArc xlink:type="arc" order="266.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_DebtInstrumentFaceAmount_2199" />
    <link:definitionArc xlink:type="arc" order="196.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionAxis_2110" xlink:to="us-gaap_RelatedPartyTransactionDomain_2111" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_TaxesOther_2024" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_TaxesPayableCurrentAndNoncurrent_2025" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_AdditionalPaidInCapitalCommonStock_2026" />
    <link:definitionArc xlink:type="arc" order="19.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_OtherLiabilities_2027" />
    <link:definitionArc xlink:type="arc" order="21.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="pfsa_PercentageOfPurchasePrice_2029" />
    <link:definitionArc xlink:type="arc" order="22.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="pfsa_PercentageOfAdditionalShares_2030" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_1485" xlink:to="us-gaap_ClassOfStockDomain_1486" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SummaryofSignificantAccountingPoliciesDetailsTable_1484" xlink:to="us-gaap_ClassOfWarrantOrRightAxis_1488" />
    <link:definitionArc xlink:type="arc" order="311.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit_2275" />
    <link:definitionArc xlink:type="arc" order="23.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_CommonStockSharesIssued_2031" />
    <link:definitionArc xlink:type="arc" order="24.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="pfsa_AdvanceNoticeShares_2032" />
    <link:definitionArc xlink:type="arc" order="27.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="pfsa_GrossProceeds_2034" />
    <link:definitionArc xlink:type="arc" order="28.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="pfsa_ShareIssuanceLiability_2035" />
    <link:definitionArc xlink:type="arc" order="29.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_InvestmentOwnedBalancePrincipalAmount_2036" />
    <link:definitionArc xlink:type="arc" order="30.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_PaymentsOfFinancingCosts_2038" />
    <link:definitionArc xlink:type="arc" order="31.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_AssetsFairValueAdjustment_2039" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1488" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_1489" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_StockholdersEquityReverseStockSplit_1492" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SummaryofSignificantAccountingPoliciesDetailsTable_1484" xlink:to="us-gaap_RelatedPartyTransactionAxis_2110" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateLineItems_1491" xlink:to="us-gaap_SharesIssuedPricePerShare_2023" />
    <link:definitionArc xlink:type="arc" order="211.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis_2128" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2129_default" />
    <link:definitionArc xlink:type="arc" order="459.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis_2464" xlink:to="us-gaap_DebtInstrumentNameDomain_2466_default" />
    <link:definitionArc xlink:type="arc" order="461.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RestatementAxis_2468" xlink:to="srt_RestatementDomain_2469_default" />
    <link:definitionArc xlink:type="arc" order="196.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionAxis_2110" xlink:to="us-gaap_RelatedPartyTransactionDomain_2111_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_1485" xlink:to="us-gaap_ClassOfStockDomain_1486_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1488" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_1489_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/FairValueMeasurementScheduleofFinancialInstrumentsthatareMeasuredatFairValueonaRecurringBasisDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member_1260" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AscentWarrantsMember" xlink:label="pfsa_AscentWarrantsMember_2191" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PrivatePlacementWarrantsMember" xlink:label="pfsa_PrivatePlacementWarrantsMember_1256" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_RepresentativesWarrantsMember" xlink:label="pfsa_RepresentativesWarrantsMember_1257" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_WarrantsLiabilitiesMember" xlink:label="pfsa_WarrantsLiabilitiesMember_1258" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis_1255" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfFinancialInstrumentsThatAreMeasuredAtFairValueOnARecurringBasisAbstract" xlink:label="pfsa_ScheduleOfFinancialInstrumentsThatAreMeasuredAtFairValueOnARecurringBasisAbstract_1253" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativesFairValueLineItems" xlink:label="us-gaap_DerivativesFairValueLineItems_1261" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstanding" xlink:label="us-gaap_WarrantsAndRightsOutstanding_1262" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAdjustmentOfWarrants" xlink:label="us-gaap_FairValueAdjustmentOfWarrants_1263" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValuesDerivativesBalanceSheetLocationByDerivativeContractTypeByHedgingDesignationTable" xlink:label="us-gaap_FairValuesDerivativesBalanceSheetLocationByDerivativeContractTypeByHedgingDesignationTable_1254" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_WarrantIssued" xlink:label="pfsa_WarrantIssued_2195" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ReclassificationOfWarrantLiabilityToAdditionalPaidinCapital" xlink:label="pfsa_ReclassificationOfWarrantLiabilityToAdditionalPaidinCapital_2196" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_1997" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1998" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis_1259" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_1997_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1998_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1998" xlink:to="us-gaap_FairValueInputsLevel3Member_1260" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1997" xlink:to="pfsa_AscentWarrantsMember_2191" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1997" xlink:to="pfsa_PrivatePlacementWarrantsMember_1256" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1997" xlink:to="pfsa_RepresentativesWarrantsMember_1257" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1997" xlink:to="pfsa_WarrantsLiabilitiesMember_1258" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValuesDerivativesBalanceSheetLocationByDerivativeContractTypeByHedgingDesignationTable_1254" xlink:to="us-gaap_ClassOfWarrantOrRightAxis_1255" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DerivativesFairValueLineItems_1261" xlink:to="us-gaap_WarrantsAndRightsOutstanding_1262" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DerivativesFairValueLineItems_1261" xlink:to="us-gaap_FairValueAdjustmentOfWarrants_1263" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_DerivativesFairValueLineItems_1261" xlink:to="us-gaap_FairValuesDerivativesBalanceSheetLocationByDerivativeContractTypeByHedgingDesignationTable_1254" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DerivativesFairValueLineItems_1261" xlink:to="pfsa_WarrantIssued_2195" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DerivativesFairValueLineItems_1261" xlink:to="pfsa_ReclassificationOfWarrantLiabilityToAdditionalPaidinCapital_2196" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1255" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_1997" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis_1259" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1998" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValuesDerivativesBalanceSheetLocationByDerivativeContractTypeByHedgingDesignationTable_1254" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis_1259" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1255" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_1997_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis_1259" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1998_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/DebtDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AprilTwentyTwentySixMember" xlink:label="pfsa_AprilTwentyTwentySixMember_2217" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PIPESubscriptionAgreementMember" xlink:label="pfsa_PIPESubscriptionAgreementMember_2219" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeniorNoteMember" xlink:label="pfsa_SeniorNoteMember_1527" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PPPLoanMember" xlink:label="pfsa_PPPLoanMember_1529" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember_1535" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FinancingArrangementMember" xlink:label="pfsa_FinancingArrangementMember_2506" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_OutstandingBorrowingsMember" xlink:label="pfsa_OutstandingBorrowingsMember_2510" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TaslyConvertibleNoteMember" xlink:label="pfsa_TaslyConvertibleNoteMember_1539" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2042" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeriesANon-VotingPreferredStockMember" xlink:label="pfsa_SeriesANon-VotingPreferredStockMember_2390" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LoansPayableMember" xlink:label="us-gaap_LoansPayableMember_1522" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SeriesAPreferredStockMember" xlink:label="us-gaap_SeriesAPreferredStockMember_2389" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AscentPIPENotesMember" xlink:label="pfsa_AscentPIPENotesMember_1538" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_1517" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember_1518" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember_1519" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PromissoryNotesMember" xlink:label="pfsa_PromissoryNotesMember_1523" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TaslyConvertibleDebtRelatedPartyAtFairValueMember" xlink:label="pfsa_TaslyConvertibleDebtRelatedPartyAtFairValueMember_1524" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleDebtMember" xlink:label="us-gaap_ConvertibleDebtMember_1525" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleNotesPayableMember" xlink:label="us-gaap_ConvertibleNotesPayableMember_1526" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConvertiblePromissoryNoteRelatedPartyMember" xlink:label="pfsa_ConvertiblePromissoryNoteRelatedPartyMember_2453" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_JuniorConvertibleNotesMember" xlink:label="pfsa_JuniorConvertibleNotesMember_2486" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuedNotesMember" xlink:label="pfsa_IssuedNotesMember_2489" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyMember" xlink:label="us-gaap_RelatedPartyMember_1532" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LoansPayableMember" xlink:label="us-gaap_LoansPayableMember_1545" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember_2048" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardDateAxis" xlink:label="us-gaap_AwardDateAxis_2215" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardDateDomain" xlink:label="us-gaap_AwardDateDomain_2216" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConversionPricePercentage" xlink:label="pfsa_ConversionPricePercentage_2218" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TradingDays" xlink:label="pfsa_TradingDays_2222" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_WarrantExpense" xlink:label="pfsa_WarrantExpense_2178" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_BeneficialOwnershipLimitationPercentage" xlink:label="pfsa_BeneficialOwnershipLimitationPercentage_2132" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_2133" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis_2137" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis_1536" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtConversionLineItems" xlink:label="us-gaap_DebtConversionLineItems_1546" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DebtExtinguishmentOnLoss" xlink:label="pfsa_DebtExtinguishmentOnLoss_2507" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FinancingCosts" xlink:label="pfsa_FinancingCosts_2174" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentInterestRateDuringPeriod" xlink:label="us-gaap_DebtInstrumentInterestRateDuringPeriod_1548" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_CashPayments" xlink:label="pfsa_CashPayments_1549" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentInterestRateIncreaseDecrease" xlink:label="us-gaap_DebtInstrumentInterestRateIncreaseDecrease_1551" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentMaturityDate" xlink:label="us-gaap_DebtInstrumentMaturityDate_1561" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding_2045" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ContractualObligation" xlink:label="us-gaap_ContractualObligation_1578" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetRetirementObligation" xlink:label="us-gaap_AssetRetirementObligation_1580" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IncrementalFairValue" xlink:label="pfsa_IncrementalFairValue_2372" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AggregatePayableAmount" xlink:label="pfsa_AggregatePayableAmount_2383" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_2388" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtTypeAxis" xlink:label="us-gaap_ShortTermDebtTypeAxis_1520" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtTypeDomain" xlink:label="us-gaap_ShortTermDebtTypeDomain_1521" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InvestmentInterestRate" xlink:label="us-gaap_InvestmentInterestRate_1562" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantExercisePriceIncrease" xlink:label="us-gaap_WarrantExercisePriceIncrease_1568" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOfDebt" xlink:label="us-gaap_ProceedsFromIssuanceOfDebt_1576" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShorttermDebtFairValue" xlink:label="us-gaap_ShorttermDebtFairValue_1556" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherLoansPayable" xlink:label="us-gaap_OtherLoansPayable_1557" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DepositLiabilitiesAccruedInterest" xlink:label="us-gaap_DepositLiabilitiesAccruedInterest_1558" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermBankLoansAndNotesPayable" xlink:label="us-gaap_ShortTermBankLoansAndNotesPayable_1559" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtPercentageBearingFixedInterestRate" xlink:label="us-gaap_ShortTermDebtPercentageBearingFixedInterestRate_1560" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtInterestRateIncrease" xlink:label="us-gaap_ShortTermDebtInterestRateIncrease_1550" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromConvertibleDebt" xlink:label="us-gaap_ProceedsFromConvertibleDebt_2380" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockConversionBasis" xlink:label="us-gaap_PreferredStockConversionBasis_2385" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="us-gaap_PreferredStockParOrStatedValuePerShare_2386" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_2387" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1537" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_2138" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentsAbstract" xlink:label="us-gaap_DebtInstrumentsAbstract_1514" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DebtDetailsTable" xlink:label="pfsa_DebtDetailsTable_1515" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis_1516" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentIssuedPrincipal" xlink:label="us-gaap_DebtInstrumentIssuedPrincipal_1553" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueOfCommonStock" xlink:label="pfsa_FairValueOfCommonStock_1555" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockConvertibleConversionPriceDecrease" xlink:label="us-gaap_CommonStockConvertibleConversionPriceDecrease_1563" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleDebt" xlink:label="us-gaap_ConvertibleDebt_1564" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleNotesPayableCurrent" xlink:label="us-gaap_ConvertibleNotesPayableCurrent_1567" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleNotesPayable" xlink:label="us-gaap_ConvertibleNotesPayable_1570" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConversionOfStockSharesConverted1" xlink:label="us-gaap_ConversionOfStockSharesConverted1_1572" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalStockSplit" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalStockSplit_1573" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AggregateCashProceeds" xlink:label="pfsa_AggregateCashProceeds_2401" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="us-gaap_DebtInstrumentFaceAmount_2040" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtConversionOriginalDebtInterestRateOfDebt" xlink:label="us-gaap_DebtConversionOriginalDebtInterestRateOfDebt_2053" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_BusinessDays" xlink:label="pfsa_BusinessDays_2054" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestExpenseDebt" xlink:label="us-gaap_InterestExpenseDebt_1571" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GainsLossesOnExtinguishmentOfDebt" xlink:label="us-gaap_GainsLossesOnExtinguishmentOfDebt_1577" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues_2416" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RepaymentsOfShortTermDebt" xlink:label="us-gaap_RepaymentsOfShortTermDebt_2051" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1530" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_1531" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis_1533" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain_1534" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InvestmentOwnedBalancePrincipalAmount" xlink:label="us-gaap_InvestmentOwnedBalancePrincipalAmount_2505" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentUnusedBorrowingCapacityAmount" xlink:label="us-gaap_DebtInstrumentUnusedBorrowingCapacityAmount_2509" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LongtermDebtTypeAxis" xlink:label="us-gaap_LongtermDebtTypeAxis_1543" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain_1544" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis_2041" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesIssuedPricePerShare" xlink:label="us-gaap_SharesIssuedPricePerShare_2046" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GeneralInsuranceExpense" xlink:label="us-gaap_GeneralInsuranceExpense_1579" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2042_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_1517_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardDateDomain" xlink:label="us-gaap_AwardDateDomain_2216_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_2388_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtTypeDomain" xlink:label="us-gaap_ShortTermDebtTypeDomain_1521_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1537_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_2138_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_1531_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain_1534_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LongtermDebtTypeDomain" xlink:label="us-gaap_LongtermDebtTypeDomain_1544_default" />
    <link:definitionArc xlink:type="arc" order="280.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AwardDateDomain_2216" xlink:to="pfsa_AprilTwentyTwentySixMember_2217" />
    <link:definitionArc xlink:type="arc" order="139.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2042" xlink:to="pfsa_PIPESubscriptionAgreementMember_2219" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="pfsa_SeniorNoteMember_1527" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="pfsa_PPPLoanMember_1529" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain_1534" xlink:to="us-gaap_CommonStockMember_1535" />
    <link:definitionArc xlink:type="arc" order="492.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1537" xlink:to="pfsa_FinancingArrangementMember_2506" />
    <link:definitionArc xlink:type="arc" order="496.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1537" xlink:to="pfsa_OutstandingBorrowingsMember_2510" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1537" xlink:to="pfsa_TaslyConvertibleNoteMember_1539" />
    <link:definitionArc xlink:type="arc" order="136.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis_2041" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2042" />
    <link:definitionArc xlink:type="arc" order="399.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_2388" xlink:to="pfsa_SeriesANon-VotingPreferredStockMember_2390" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="us-gaap_LoansPayableMember_1522" />
    <link:definitionArc xlink:type="arc" order="398.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_2388" xlink:to="us-gaap_SeriesAPreferredStockMember_2389" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1537" xlink:to="pfsa_AscentPIPENotesMember_1538" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis_1516" xlink:to="srt_RangeMember_1517" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_1517" xlink:to="srt_MinimumMember_1518" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_1517" xlink:to="srt_MaximumMember_1519" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="pfsa_PromissoryNotesMember_1523" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="pfsa_TaslyConvertibleDebtRelatedPartyAtFairValueMember_1524" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="us-gaap_ConvertibleDebtMember_1525" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="us-gaap_ConvertibleNotesPayableMember_1526" />
    <link:definitionArc xlink:type="arc" order="477.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="pfsa_ConvertiblePromissoryNoteRelatedPartyMember_2453" />
    <link:definitionArc xlink:type="arc" order="476.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="pfsa_JuniorConvertibleNotesMember_2486" />
    <link:definitionArc xlink:type="arc" order="480.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_1521" xlink:to="pfsa_IssuedNotesMember_2489" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain_1531" xlink:to="us-gaap_RelatedPartyMember_1532" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LongtermDebtTypeDomain_1544" xlink:to="us-gaap_LoansPayableMember_1545" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_2138" xlink:to="us-gaap_WarrantMember_2048" />
    <link:definitionArc xlink:type="arc" order="21.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="us-gaap_AwardDateAxis_2215" />
    <link:definitionArc xlink:type="arc" order="279.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardDateAxis_2215" xlink:to="us-gaap_AwardDateDomain_2216" />
    <link:definitionArc xlink:type="arc" order="281.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_ConversionPricePercentage_2218" />
    <link:definitionArc xlink:type="arc" order="282.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_TradingDays_2222" />
    <link:definitionArc xlink:type="arc" order="252.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_WarrantExpense_2178" />
    <link:definitionArc xlink:type="arc" order="53.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_BeneficialOwnershipLimitationPercentage_2132" />
    <link:definitionArc xlink:type="arc" order="46.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_2133" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="us-gaap_ClassOfWarrantOrRightAxis_2137" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="us-gaap_DebtInstrumentAxis_1536" />
    <link:definitionArc xlink:type="arc" order="493.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_DebtExtinguishmentOnLoss_2507" />
    <link:definitionArc xlink:type="arc" order="248.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_FinancingCosts_2174" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_DebtInstrumentInterestRateDuringPeriod_1548" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_CashPayments_1549" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_DebtInstrumentInterestRateIncreaseDecrease_1551" />
    <link:definitionArc xlink:type="arc" order="24.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_DebtInstrumentMaturityDate_1561" />
    <link:definitionArc xlink:type="arc" order="45.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding_2045" />
    <link:definitionArc xlink:type="arc" order="41.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ContractualObligation_1578" />
    <link:definitionArc xlink:type="arc" order="43.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_AssetRetirementObligation_1580" />
    <link:definitionArc xlink:type="arc" order="382.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_IncrementalFairValue_2372" />
    <link:definitionArc xlink:type="arc" order="392.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_AggregatePayableAmount_2383" />
    <link:definitionArc xlink:type="arc" order="397.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_2387" xlink:to="us-gaap_ClassOfStockDomain_2388" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="us-gaap_ShortTermDebtTypeAxis_1520" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ShortTermDebtTypeAxis_1520" xlink:to="us-gaap_ShortTermDebtTypeDomain_1521" />
    <link:definitionArc xlink:type="arc" order="25.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_InvestmentInterestRate_1562" />
    <link:definitionArc xlink:type="arc" order="31.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_WarrantExercisePriceIncrease_1568" />
    <link:definitionArc xlink:type="arc" order="39.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ProceedsFromIssuanceOfDebt_1576" />
    <link:definitionArc xlink:type="arc" order="19.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ShorttermDebtFairValue_1556" />
    <link:definitionArc xlink:type="arc" order="20.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_OtherLoansPayable_1557" />
    <link:definitionArc xlink:type="arc" order="21.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_DepositLiabilitiesAccruedInterest_1558" />
    <link:definitionArc xlink:type="arc" order="22.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ShortTermBankLoansAndNotesPayable_1559" />
    <link:definitionArc xlink:type="arc" order="23.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ShortTermDebtPercentageBearingFixedInterestRate_1560" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ShortTermDebtInterestRateIncrease_1550" />
    <link:definitionArc xlink:type="arc" order="389.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ProceedsFromConvertibleDebt_2380" />
    <link:definitionArc xlink:type="arc" order="394.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_PreferredStockConversionBasis_2385" />
    <link:definitionArc xlink:type="arc" order="395.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_PreferredStockParOrStatedValuePerShare_2386" />
    <link:definitionArc xlink:type="arc" order="22.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="us-gaap_StatementClassOfStockAxis_2387" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis_1536" xlink:to="us-gaap_DebtInstrumentNameDomain_1537" />
    <link:definitionArc xlink:type="arc" order="216.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_2137" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_2138" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_DebtDetailsTable_1515" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="srt_RangeAxis_1516" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_DebtInstrumentIssuedPrincipal_1553" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_FairValueOfCommonStock_1555" />
    <link:definitionArc xlink:type="arc" order="26.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_CommonStockConvertibleConversionPriceDecrease_1563" />
    <link:definitionArc xlink:type="arc" order="27.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ConvertibleDebt_1564" />
    <link:definitionArc xlink:type="arc" order="30.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ConvertibleNotesPayableCurrent_1567" />
    <link:definitionArc xlink:type="arc" order="33.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ConvertibleNotesPayable_1570" />
    <link:definitionArc xlink:type="arc" order="35.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_ConversionOfStockSharesConverted1_1572" />
    <link:definitionArc xlink:type="arc" order="36.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalStockSplit_1573" />
    <link:definitionArc xlink:type="arc" order="410.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_AggregateCashProceeds_2401" />
    <link:definitionArc xlink:type="arc" order="44.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_DebtInstrumentFaceAmount_2040" />
    <link:definitionArc xlink:type="arc" order="50.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_DebtConversionOriginalDebtInterestRateOfDebt_2053" />
    <link:definitionArc xlink:type="arc" order="51.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="pfsa_BusinessDays_2054" />
    <link:definitionArc xlink:type="arc" order="34.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_InterestExpenseDebt_1571" />
    <link:definitionArc xlink:type="arc" order="40.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_GainsLossesOnExtinguishmentOfDebt_1577" />
    <link:definitionArc xlink:type="arc" order="423.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues_2416" />
    <link:definitionArc xlink:type="arc" order="49.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_RepaymentsOfShortTermDebt_2051" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1530" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1530" xlink:to="us-gaap_RelatedPartyDomain_1531" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="us-gaap_StatementEquityComponentsAxis_1533" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis_1533" xlink:to="us-gaap_EquityComponentDomain_1534" />
    <link:definitionArc xlink:type="arc" order="491.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_InvestmentOwnedBalancePrincipalAmount_2505" />
    <link:definitionArc xlink:type="arc" order="495.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_DebtInstrumentUnusedBorrowingCapacityAmount_2509" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="us-gaap_LongtermDebtTypeAxis_1543" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LongtermDebtTypeAxis_1543" xlink:to="us-gaap_LongtermDebtTypeDomain_1544" />
    <link:definitionArc xlink:type="arc" order="19.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_DebtDetailsTable_1515" xlink:to="us-gaap_TypeOfArrangementAxis_2041" />
    <link:definitionArc xlink:type="arc" order="47.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_SharesIssuedPricePerShare_2046" />
    <link:definitionArc xlink:type="arc" order="42.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtConversionLineItems_1546" xlink:to="us-gaap_GeneralInsuranceExpense_1579" />
    <link:definitionArc xlink:type="arc" order="136.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis_2041" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2042_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis_1516" xlink:to="srt_RangeMember_1517_default" />
    <link:definitionArc xlink:type="arc" order="279.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardDateAxis_2215" xlink:to="us-gaap_AwardDateDomain_2216_default" />
    <link:definitionArc xlink:type="arc" order="397.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_2387" xlink:to="us-gaap_ClassOfStockDomain_2388_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ShortTermDebtTypeAxis_1520" xlink:to="us-gaap_ShortTermDebtTypeDomain_1521_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis_1536" xlink:to="us-gaap_DebtInstrumentNameDomain_1537_default" />
    <link:definitionArc xlink:type="arc" order="216.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_2137" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_2138_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1530" xlink:to="us-gaap_RelatedPartyDomain_1531_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis_1533" xlink:to="us-gaap_EquityComponentDomain_1534_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LongtermDebtTypeAxis_1543" xlink:to="us-gaap_LongtermDebtTypeDomain_1544_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/FairValueMeasurementScheduleofInputsintotheMonteCarloSimulationModelfortheWarrantLiabilitiesandConvertibleLoansPayableDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputExercisePriceMember" xlink:label="us-gaap_MeasurementInputExercisePriceMember_1248" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueOfCommonStockMember" xlink:label="pfsa_FairValueOfCommonStockMember_1249" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_WarrantLiabilitiesMember" xlink:label="pfsa_WarrantLiabilitiesMember_1242" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LoansPayablesMember" xlink:label="pfsa_LoansPayablesMember_1243" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:label="us-gaap_MeasurementInputRiskFreeInterestRateMember_1245" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputExpectedTermMember" xlink:label="us-gaap_MeasurementInputExpectedTermMember_1246" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputPriceVolatilityMember" xlink:label="us-gaap_MeasurementInputPriceVolatilityMember_1247" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2438" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember_2439" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember_2440" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_1250" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstandingMeasurementInput" xlink:label="us-gaap_WarrantsAndRightsOutstandingMeasurementInput_1251" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfInputsIntoTheMonteCarloSimulationModelForTheWarrantLiabilitiesAbstract" xlink:label="pfsa_ScheduleOfInputsIntoTheMonteCarloSimulationModelForTheWarrantLiabilitiesAbstract_1239" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_1240" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis_1241" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="us-gaap_MeasurementInputTypeAxis_1244" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1995" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain_1996" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis_2437" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2438_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1995_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain_1996_default" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_1996" xlink:to="us-gaap_MeasurementInputExercisePriceMember_1248" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_1996" xlink:to="pfsa_FairValueOfCommonStockMember_1249" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1995" xlink:to="pfsa_WarrantLiabilitiesMember_1242" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1995" xlink:to="pfsa_LoansPayablesMember_1243" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_1996" xlink:to="us-gaap_MeasurementInputRiskFreeInterestRateMember_1245" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_1996" xlink:to="us-gaap_MeasurementInputExpectedTermMember_1246" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_1996" xlink:to="us-gaap_MeasurementInputPriceVolatilityMember_1247" />
    <link:definitionArc xlink:type="arc" order="443.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis_2437" xlink:to="srt_RangeMember_2438" />
    <link:definitionArc xlink:type="arc" order="444.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2438" xlink:to="srt_MaximumMember_2439" />
    <link:definitionArc xlink:type="arc" order="445.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2438" xlink:to="srt_MinimumMember_2440" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_1250" xlink:to="us-gaap_WarrantsAndRightsOutstandingMeasurementInput_1251" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_1250" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_1240" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_1240" xlink:to="us-gaap_FinancialInstrumentAxis_1241" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_1240" xlink:to="us-gaap_MeasurementInputTypeAxis_1244" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FinancialInstrumentAxis_1241" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1995" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_MeasurementInputTypeAxis_1244" xlink:to="us-gaap_MeasurementInputTypeDomain_1996" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_1240" xlink:to="srt_RangeAxis_2437" />
    <link:definitionArc xlink:type="arc" order="443.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis_2437" xlink:to="srt_RangeMember_2438_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FinancialInstrumentAxis_1241" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_1995_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_MeasurementInputTypeAxis_1244" xlink:to="us-gaap_MeasurementInputTypeDomain_1996_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/FairValueMeasurementScheduleofFinancialAssetsandLiabilitiesMeasuredatFairValueonaRecurringBasisDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_RepresentativesWarrantsMember" xlink:label="pfsa_RepresentativesWarrantsMember_1224" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member_1227" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel2Member" xlink:label="us-gaap_FairValueInputsLevel2Member_1228" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member_1229" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PrivatePlacementWarrantsMember" xlink:label="pfsa_PrivatePlacementWarrantsMember_1223" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsRecurringMember" xlink:label="us-gaap_FairValueMeasurementsRecurringMember_2116" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis_1225" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1226" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsAbstract" xlink:label="us-gaap_AssetsAbstract_1236" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CryptoAssetFairValueCurrent" xlink:label="us-gaap_CryptoAssetFairValueCurrent_1237" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsLineItems" xlink:label="pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsLineItems_1230" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesAbstract" xlink:label="us-gaap_LiabilitiesAbstract_1231" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleDebtFairValueDisclosures" xlink:label="us-gaap_ConvertibleDebtFairValueDisclosures_1232" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleDebtNoncurrent" xlink:label="us-gaap_ConvertibleDebtNoncurrent_1233" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesFairValueDisclosure" xlink:label="us-gaap_LiabilitiesFairValueDisclosure_1235" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfCompanySFinancialAssetsAndLiabilitiesMeasuredAtFairValueAbstract" xlink:label="pfsa_ScheduleOfCompanySFinancialAssetsAndLiabilitiesMeasuredAtFairValueAbstract_1219" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsTable" xlink:label="pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsTable_1220" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis_1221" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_1222" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_WarrantLiabilities" xlink:label="pfsa_WarrantLiabilities_2113" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByMeasurementFrequencyAxis" xlink:label="us-gaap_FairValueByMeasurementFrequencyAxis_2114" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain_2115" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1226_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_1222_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain_2115_default" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1222" xlink:to="pfsa_RepresentativesWarrantsMember_1224" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1226" xlink:to="us-gaap_FairValueInputsLevel1Member_1227" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1226" xlink:to="us-gaap_FairValueInputsLevel2Member_1228" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1226" xlink:to="us-gaap_FairValueInputsLevel3Member_1229" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1222" xlink:to="pfsa_PrivatePlacementWarrantsMember_1223" />
    <link:definitionArc xlink:type="arc" order="200.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementFrequencyDomain_2115" xlink:to="us-gaap_FairValueMeasurementsRecurringMember_2116" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsTable_1220" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis_1225" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis_1225" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1226" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsLineItems_1230" xlink:to="us-gaap_AssetsAbstract_1236" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract_1236" xlink:to="us-gaap_CryptoAssetFairValueCurrent_1237" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsLineItems_1230" xlink:to="us-gaap_LiabilitiesAbstract_1231" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAbstract_1231" xlink:to="us-gaap_ConvertibleDebtFairValueDisclosures_1232" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAbstract_1231" xlink:to="us-gaap_ConvertibleDebtNoncurrent_1233" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAbstract_1231" xlink:to="us-gaap_LiabilitiesFairValueDisclosure_1235" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsLineItems_1230" xlink:to="pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsTable_1220" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsTable_1220" xlink:to="us-gaap_ClassOfWarrantOrRightAxis_1221" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1221" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_1222" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAbstract_1231" xlink:to="pfsa_WarrantLiabilities_2113" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_FairValueMeasurementScheduleofCompanysFinancialAssetsandLiabilitiesMeasuredatFairValueDetailsTable_1220" xlink:to="us-gaap_FairValueByMeasurementFrequencyAxis_2114" />
    <link:definitionArc xlink:type="arc" order="199.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis_2114" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain_2115" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis_1225" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_1226_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1221" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_1222_default" />
    <link:definitionArc xlink:type="arc" order="199.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis_2114" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain_2115_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/FairValueMeasurementDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember_1502" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueDisclosuresAbstract" xlink:label="us-gaap_FairValueDisclosuresAbstract_1498" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueMeasurementDetailsTable" xlink:label="pfsa_FairValueMeasurementDetailsTable_1499" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_1500" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConversionValues" xlink:label="pfsa_ConversionValues_2147" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstanding" xlink:label="us-gaap_WarrantsAndRightsOutstanding_2500" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ContractualTerm" xlink:label="pfsa_ContractualTerm_2172" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CryptoAssetCost" xlink:label="us-gaap_CryptoAssetCost_1510" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CryptoAssetFairValue" xlink:label="us-gaap_CryptoAssetFairValue_1511" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CryptoAssetMining" xlink:label="us-gaap_CryptoAssetMining_1512" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1501" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice_1507" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SoldBitcoins" xlink:label="pfsa_SoldBitcoins_1509" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_NumberOfShares" xlink:label="pfsa_NumberOfShares_2049" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1501_default" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1501" xlink:to="us-gaap_CommonStockMember_1502" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="pfsa_FairValueMeasurementDetailsTable_1499" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_FairValueMeasurementDetailsTable_1499" xlink:to="us-gaap_StatementClassOfStockAxis_1500" />
    <link:definitionArc xlink:type="arc" order="222.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="pfsa_ConversionValues_2147" />
    <link:definitionArc xlink:type="arc" order="487.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="us-gaap_WarrantsAndRightsOutstanding_2500" />
    <link:definitionArc xlink:type="arc" order="246.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="pfsa_ContractualTerm_2172" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="us-gaap_CryptoAssetCost_1510" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="us-gaap_CryptoAssetFairValue_1511" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="us-gaap_CryptoAssetMining_1512" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_1500" xlink:to="us-gaap_ClassOfStockDomain_1501" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="us-gaap_SharePrice_1507" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="pfsa_SoldBitcoins_1509" />
    <link:definitionArc xlink:type="arc" order="142.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_1506" xlink:to="pfsa_NumberOfShares_2049" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_1500" xlink:to="us-gaap_ClassOfStockDomain_1501_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/FairValueMeasurementScheduleofChangesintheFairValueoftheCompanysLevel3LoansPayableDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member_1280" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DebtExtinguishmentOfFairValue" xlink:label="pfsa_DebtExtinguishmentOfFairValue_2501" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueOptionChangesInFairValueGainLoss1" xlink:label="us-gaap_FairValueOptionChangesInFairValueGainLoss1_1285" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ProceedsReceived" xlink:label="pfsa_ProceedsReceived_2047" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueMeasurementScheduleofChangesintheFairValueoftheCompanysLevel3RelatedPartyLoanPayableDetailsTable" xlink:label="pfsa_FairValueMeasurementScheduleofChangesintheFairValueoftheCompanysLevel3RelatedPartyLoanPayableDetailsTable_1278" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis_1279" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems" xlink:label="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1281" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LoansPayableRelatedPartyAtFairValue" xlink:label="pfsa_LoansPayableRelatedPartyAtFairValue_1282" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RepaymentsOfDebt" xlink:label="us-gaap_RepaymentsOfDebt_1283" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_StockIssuedDuringPeriodValueIssuanceOfSharesInConnectionWithTheConversionOfThePIPENote" xlink:label="pfsa_StockIssuedDuringPeriodValueIssuanceOfSharesInConnectionWithTheConversionOfThePIPENote_1284" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2000" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfChangesInTheFairValueOfTheCompanySLevel3RelatedPartyLoanPayableAbstract" xlink:label="pfsa_ScheduleOfChangesInTheFairValueOfTheCompanySLevel3RelatedPartyLoanPayableAbstract_1277" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2000_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2000" xlink:to="us-gaap_FairValueInputsLevel3Member_1280" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1281" xlink:to="pfsa_DebtExtinguishmentOfFairValue_2501" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1281" xlink:to="us-gaap_FairValueOptionChangesInFairValueGainLoss1_1285" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1281" xlink:to="pfsa_ProceedsReceived_2047" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1281" xlink:to="pfsa_FairValueMeasurementScheduleofChangesintheFairValueoftheCompanysLevel3RelatedPartyLoanPayableDetailsTable_1278" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_FairValueMeasurementScheduleofChangesintheFairValueoftheCompanysLevel3RelatedPartyLoanPayableDetailsTable_1278" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis_1279" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1281" xlink:to="pfsa_LoansPayableRelatedPartyAtFairValue_1282" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1281" xlink:to="us-gaap_RepaymentsOfDebt_1283" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_1281" xlink:to="pfsa_StockIssuedDuringPeriodValueIssuanceOfSharesInConnectionWithTheConversionOfThePIPENote_1284" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis_1279" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2000" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis_1279" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2000_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/SubsequentEventsDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2448" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember_1673" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1676" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PIPESubscriptionAgreementMember" xlink:label="pfsa_PIPESubscriptionAgreementMember_1678" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PromissoryNoteMember" xlink:label="pfsa_PromissoryNoteMember_1688" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ScenarioForecastMember" xlink:label="srt_ScenarioForecastMember_1694" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember_1685" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConversionPriceOfPerShare" xlink:label="pfsa_ConversionPriceOfPerShare_1699" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockValueOutstanding" xlink:label="us-gaap_CommonStockValueOutstanding_1700" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_OriginalDiscountAmount" xlink:label="pfsa_OriginalDiscountAmount_2475" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="us-gaap_DebtInstrumentInterestRateStatedPercentage_2221" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SubsequentEventsDetailsTable" xlink:label="pfsa_SubsequentEventsDetailsTable_1670" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConversionPricePercentage" xlink:label="pfsa_ConversionPricePercentage_2396" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ContractualFloorPrice" xlink:label="pfsa_ContractualFloorPrice_2398" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtConversionConvertedInstrumentAmount1" xlink:label="us-gaap_DebtConversionConvertedInstrumentAmount1_2517" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtConversionConvertedInstrumentSharesIssued1" xlink:label="us-gaap_DebtConversionConvertedInstrumentSharesIssued1_2518" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="us-gaap_DebtInstrumentFaceAmount_1702" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis_1686" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1687" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_CashProceeds" xlink:label="pfsa_CashProceeds_2403" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ReservationOfMaximumAggregateNumberOfShares" xlink:label="pfsa_ReservationOfMaximumAggregateNumberOfShares_2407" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentIssuedPrincipal" xlink:label="us-gaap_DebtInstrumentIssuedPrincipal_1704" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentConvertibleConversionPrice1" xlink:label="us-gaap_DebtInstrumentConvertibleConversionPrice1_2514" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1672" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_StatementScenarioAxis" xlink:label="srt_StatementScenarioAxis_1692" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ScenarioUnspecifiedDomain" xlink:label="srt_ScenarioUnspecifiedDomain_1693" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventLineItems" xlink:label="us-gaap_SubsequentEventLineItems_1695" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis_1683" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain_1684" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventsAbstract" xlink:label="us-gaap_SubsequentEventsAbstract_1669" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_1671" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis_1675" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1676_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1687_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1672_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ScenarioUnspecifiedDomain" xlink:label="srt_ScenarioUnspecifiedDomain_1693_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain_1684_default" />
    <link:definitionArc xlink:type="arc" order="233.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1672" xlink:to="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2448" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1672" xlink:to="us-gaap_CommonStockMember_1673" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis_1675" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1676" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1676" xlink:to="pfsa_PIPESubscriptionAgreementMember_1678" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1687" xlink:to="pfsa_PromissoryNoteMember_1688" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ScenarioUnspecifiedDomain_1693" xlink:to="srt_ScenarioForecastMember_1694" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain_1684" xlink:to="us-gaap_SubsequentEventMember_1685" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="pfsa_ConversionPriceOfPerShare_1699" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="us-gaap_CommonStockValueOutstanding_1700" />
    <link:definitionArc xlink:type="arc" order="466.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="pfsa_OriginalDiscountAmount_2475" />
    <link:definitionArc xlink:type="arc" order="262.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="us-gaap_DebtInstrumentInterestRateStatedPercentage_2221" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="pfsa_SubsequentEventsDetailsTable_1670" />
    <link:definitionArc xlink:type="arc" order="405.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="pfsa_ConversionPricePercentage_2396" />
    <link:definitionArc xlink:type="arc" order="407.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="pfsa_ContractualFloorPrice_2398" />
    <link:definitionArc xlink:type="arc" order="503.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="us-gaap_DebtConversionConvertedInstrumentAmount1_2517" />
    <link:definitionArc xlink:type="arc" order="504.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="us-gaap_DebtConversionConvertedInstrumentSharesIssued1_2518" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="us-gaap_DebtInstrumentFaceAmount_1702" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SubsequentEventsDetailsTable_1670" xlink:to="us-gaap_DebtInstrumentAxis_1686" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis_1686" xlink:to="us-gaap_DebtInstrumentNameDomain_1687" />
    <link:definitionArc xlink:type="arc" order="412.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="pfsa_CashProceeds_2403" />
    <link:definitionArc xlink:type="arc" order="414.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="pfsa_ReservationOfMaximumAggregateNumberOfShares_2407" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="us-gaap_DebtInstrumentIssuedPrincipal_1704" />
    <link:definitionArc xlink:type="arc" order="500.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems_1695" xlink:to="us-gaap_DebtInstrumentConvertibleConversionPrice1_2514" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_1671" xlink:to="us-gaap_ClassOfStockDomain_1672" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SubsequentEventsDetailsTable_1670" xlink:to="srt_StatementScenarioAxis_1692" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_StatementScenarioAxis_1692" xlink:to="srt_ScenarioUnspecifiedDomain_1693" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SubsequentEventsDetailsTable_1670" xlink:to="us-gaap_SubsequentEventTypeAxis_1683" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis_1683" xlink:to="us-gaap_SubsequentEventTypeDomain_1684" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SubsequentEventsDetailsTable_1670" xlink:to="us-gaap_StatementClassOfStockAxis_1671" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SubsequentEventsDetailsTable_1670" xlink:to="us-gaap_TypeOfArrangementAxis_1675" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis_1675" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1676_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis_1686" xlink:to="us-gaap_DebtInstrumentNameDomain_1687_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_1671" xlink:to="us-gaap_ClassOfStockDomain_1672_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_StatementScenarioAxis_1692" xlink:to="srt_ScenarioUnspecifiedDomain_1693_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsequentEventTypeAxis_1683" xlink:to="us-gaap_SubsequentEventTypeDomain_1684_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentParentheticalsDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingSegmentsMember" xlink:label="us-gaap_OperatingSegmentsMember_1454" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsDomain" xlink:label="srt_ConsolidationItemsDomain_2004" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfOtherFinancialInformationRegardingTheCompanySOperatingSegmentAbstract" xlink:label="pfsa_ScheduleOfOtherFinancialInformationRegardingTheCompanySOperatingSegmentAbstract_1451" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentParentheticalsDetailsTable" xlink:label="pfsa_SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentParentheticalsDetailsTable_1452" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsAxis" xlink:label="srt_ConsolidationItemsAxis_1453" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentReportingInformationLineItems" xlink:label="us-gaap_SegmentReportingInformationLineItems_1455" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestExpenseOther" xlink:label="us-gaap_InterestExpenseOther_1456" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LossOnExtinguishmentOfRelatedPartyAmountDebt" xlink:label="pfsa_LossOnExtinguishmentOfRelatedPartyAmountDebt_2513" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsDomain" xlink:label="srt_ConsolidationItemsDomain_2004_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ConsolidationItemsDomain_2004" xlink:to="us-gaap_OperatingSegmentsMember_1454" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ConsolidationItemsAxis_1453" xlink:to="srt_ConsolidationItemsDomain_2004" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_SegmentReportingInformationLineItems_1455" xlink:to="pfsa_SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentParentheticalsDetailsTable_1452" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentParentheticalsDetailsTable_1452" xlink:to="srt_ConsolidationItemsAxis_1453" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems_1455" xlink:to="us-gaap_InterestExpenseOther_1456" />
    <link:definitionArc xlink:type="arc" order="499.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems_1455" xlink:to="pfsa_LossOnExtinguishmentOfRelatedPartyAmountDebt_2513" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ConsolidationItemsAxis_1453" xlink:to="srt_ConsolidationItemsDomain_2004_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/CommonStockWarrantsDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2455" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PIPESubscriptionAgreementMember" xlink:label="pfsa_PIPESubscriptionAgreementMember_2456" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_WarrantAgreementMember" xlink:label="pfsa_WarrantAgreementMember_2493" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AscentWarrantsMember" xlink:label="pfsa_AscentWarrantsMember_2074" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2077" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember_2078" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember_2079" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_CommonStockWarrantsMember" xlink:label="pfsa_CommonStockWarrantsMember_2170" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PublicWarrantMember" xlink:label="pfsa_PublicWarrantMember_1619" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PrivatePlacementWarrantsMember" xlink:label="pfsa_PrivatePlacementWarrantsMember_1620" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_RepresentativesWarrantsMember" xlink:label="pfsa_RepresentativesWarrantsMember_1621" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_HCWWarrantsMember" xlink:label="pfsa_HCWWarrantsMember_1622" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_2005" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SharesIssuableUponExerciseOfWarrants" xlink:label="pfsa_SharesIssuableUponExerciseOfWarrants_2135" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis_2454" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_WarrantsToPurchaseShares" xlink:label="pfsa_WarrantsToPurchaseShares_2480" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SharesOfReverseStockSplit" xlink:label="pfsa_SharesOfReverseStockSplit_2481" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SharesOfWarrantsExercisable" xlink:label="pfsa_SharesOfWarrantsExercisable_2482" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AggregateExercisePrice" xlink:label="pfsa_AggregateExercisePrice_2485" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LoanCommitmentAssets" xlink:label="pfsa_LoanCommitmentAssets_2208" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_GainLossOnAscentWarrant" xlink:label="pfsa_GainLossOnAscentWarrant_2209" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalMarkToMarket" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalMarkToMarket_2376" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesReverseStockSplits" xlink:label="us-gaap_StockIssuedDuringPeriodSharesReverseStockSplits_2382" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueOfAscentWarrant" xlink:label="pfsa_FairValueOfAscentWarrant_2201" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueOfIssuanceCost" xlink:label="pfsa_FairValueOfIssuanceCost_2334" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_2335" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDisclosuresAbstract" xlink:label="us-gaap_ClassOfStockDisclosuresAbstract_1615" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_CommonStockWarrantsDetailsTable" xlink:label="pfsa_CommonStockWarrantsDetailsTable_1616" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis_2076" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis_1617" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockLineItems" xlink:label="us-gaap_ClassOfStockLineItems_1623" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding_1624" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2455_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2077_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_2005_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis_2454" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2455" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2455" xlink:to="pfsa_PIPESubscriptionAgreementMember_2456" />
    <link:definitionArc xlink:type="arc" order="484.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2455" xlink:to="pfsa_WarrantAgreementMember_2493" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_2005" xlink:to="pfsa_AscentWarrantsMember_2074" />
    <link:definitionArc xlink:type="arc" order="166.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis_2076" xlink:to="srt_RangeMember_2077" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2077" xlink:to="srt_MaximumMember_2078" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2077" xlink:to="srt_MinimumMember_2079" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_2005" xlink:to="pfsa_CommonStockWarrantsMember_2170" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_2005" xlink:to="pfsa_PublicWarrantMember_1619" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_2005" xlink:to="pfsa_PrivatePlacementWarrantsMember_1620" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_2005" xlink:to="pfsa_RepresentativesWarrantsMember_1621" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_2005" xlink:to="pfsa_HCWWarrantsMember_1622" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1617" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_2005" />
    <link:definitionArc xlink:type="arc" order="213.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_SharesIssuableUponExerciseOfWarrants_2135" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_CommonStockWarrantsDetailsTable_1616" xlink:to="us-gaap_TypeOfArrangementAxis_2454" />
    <link:definitionArc xlink:type="arc" order="470.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_WarrantsToPurchaseShares_2480" />
    <link:definitionArc xlink:type="arc" order="471.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_SharesOfReverseStockSplit_2481" />
    <link:definitionArc xlink:type="arc" order="472.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_SharesOfWarrantsExercisable_2482" />
    <link:definitionArc xlink:type="arc" order="475.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_AggregateExercisePrice_2485" />
    <link:definitionArc xlink:type="arc" order="275.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_LoanCommitmentAssets_2208" />
    <link:definitionArc xlink:type="arc" order="276.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_GainLossOnAscentWarrant_2209" />
    <link:definitionArc xlink:type="arc" order="386.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalMarkToMarket_2376" />
    <link:definitionArc xlink:type="arc" order="391.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="us-gaap_StockIssuedDuringPeriodSharesReverseStockSplits_2382" />
    <link:definitionArc xlink:type="arc" order="268.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_FairValueOfAscentWarrant_2201" />
    <link:definitionArc xlink:type="arc" order="353.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_FairValueOfIssuanceCost_2334" />
    <link:definitionArc xlink:type="arc" order="354.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_2335" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="pfsa_CommonStockWarrantsDetailsTable_1616" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_CommonStockWarrantsDetailsTable_1616" xlink:to="srt_RangeAxis_2076" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_CommonStockWarrantsDetailsTable_1616" xlink:to="us-gaap_ClassOfWarrantOrRightAxis_1617" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1623" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding_1624" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis_2454" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2455_default" />
    <link:definitionArc xlink:type="arc" order="166.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis_2076" xlink:to="srt_RangeMember_2077_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1617" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_2005_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/StockOptionPlanDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TwoThousandTenEquityIncentivePlanMember" xlink:label="pfsa_TwoThousandTenEquityIncentivePlanMember_1629" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ThousandTwentyFiveIncentivePlanMember" xlink:label="pfsa_ThousandTwentyFiveIncentivePlanMember_1630" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain_2006" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationAbstract" xlink:label="us-gaap_ShareBasedCompensationAbstract_1626" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_StockOptionPlanDetailsTable" xlink:label="pfsa_StockOptionPlanDetailsTable_1627" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis_1628" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DeferredCompensationArrangementWithIndividualShareBasedPaymentsLineItems" xlink:label="us-gaap_DeferredCompensationArrangementWithIndividualShareBasedPaymentsLineItems_1631" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_1632" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant_1633" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain_2006_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain_2006" xlink:to="pfsa_TwoThousandTenEquityIncentivePlanMember_1629" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain_2006" xlink:to="pfsa_ThousandTwentyFiveIncentivePlanMember_1630" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis_1628" xlink:to="us-gaap_PlanNameDomain_2006" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_DeferredCompensationArrangementWithIndividualShareBasedPaymentsLineItems_1631" xlink:to="pfsa_StockOptionPlanDetailsTable_1627" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_StockOptionPlanDetailsTable_1627" xlink:to="us-gaap_PlanNameAxis_1628" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DeferredCompensationArrangementWithIndividualShareBasedPaymentsLineItems_1631" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_1632" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DeferredCompensationArrangementWithIndividualShareBasedPaymentsLineItems_1631" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant_1633" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis_1628" xlink:to="us-gaap_PlanNameDomain_2006_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/NetLossPerShareAttributabletoCommonStockholdersScheduleofDilutedNetLossPerShareAttributabletoCommonStockholdersDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2474" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_RelatedPartyConvertibleNotesMember" xlink:label="pfsa_RelatedPartyConvertibleNotesMember_1419" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember_1421" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_OptionsToPurchaseCommonStockMember" xlink:label="pfsa_OptionsToPurchaseCommonStockMember_1422" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_EarnoutSharesMember" xlink:label="pfsa_EarnoutSharesMember_1423" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleNotesPayableMember" xlink:label="us-gaap_ConvertibleNotesPayableMember_1418" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfDilutedNetLossPerShareAttributableToCommonStockholdersAbstract" xlink:label="pfsa_ScheduleOfDilutedNetLossPerShareAttributableToCommonStockholdersAbstract_1413" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_1414" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_1415" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain_1416" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_1424" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_1425" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain_1416_default" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain_1416" xlink:to="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2474" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain_1416" xlink:to="pfsa_RelatedPartyConvertibleNotesMember_1419" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain_1416" xlink:to="us-gaap_WarrantMember_1421" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain_1416" xlink:to="pfsa_OptionsToPurchaseCommonStockMember_1422" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain_1416" xlink:to="pfsa_EarnoutSharesMember_1423" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain_1416" xlink:to="us-gaap_ConvertibleNotesPayableMember_1418" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_1424" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_1414" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_1414" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_1415" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_1415" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain_1416" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_1424" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_1425" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_1415" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain_1416_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/DebtScheduleofDebtInstrumentsandtheirChangesDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember_1303" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TaslyConvertibleDebtRelatedPartyMember" xlink:label="pfsa_TaslyConvertibleDebtRelatedPartyMember_1309" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConvertiblePromissoryNoteRelatedPartyMember" xlink:label="pfsa_ConvertiblePromissoryNoteRelatedPartyMember_1310" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeniorNoteMember" xlink:label="pfsa_SeniorNoteMember_1311" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PromissoryNotesMember" xlink:label="pfsa_PromissoryNotesMember_1312" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PPPLoanMember" xlink:label="pfsa_PPPLoanMember_1313" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DOInsuranceFinancingMember" xlink:label="pfsa_DOInsuranceFinancingMember_1314" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_JuniorConvertibleNotesMember" xlink:label="pfsa_JuniorConvertibleNotesMember_1315" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LoansPayablesInstrumentMember" xlink:label="pfsa_LoansPayablesInstrumentMember_2140" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_1301" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember_1302" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentLineItems" xlink:label="us-gaap_DebtInstrumentLineItems_1316" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConversionOfDebtToEquity" xlink:label="pfsa_ConversionOfDebtToEquity_1324" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_StatedInterest" xlink:label="pfsa_StatedInterest_1325" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AmortizationOfFinancingCostsAndDiscounts" xlink:label="us-gaap_AmortizationOfFinancingCostsAndDiscounts_1326" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAdjustmentOfWarrants" xlink:label="us-gaap_FairValueAdjustmentOfWarrants_1327" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DebtConversionPricesPerShare" xlink:label="pfsa_DebtConversionPricesPerShare_1320" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentMaturityDate" xlink:label="us-gaap_DebtInstrumentMaturityDate_1321" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DebtRepayments" xlink:label="pfsa_DebtRepayments_1322" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOfDebt" xlink:label="us-gaap_ProceedsFromIssuanceOfDebt_1323" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis_1307" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1308" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DebtForgiveness" xlink:label="pfsa_DebtForgiveness_2134" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfDebtInstrumentsAndTheirChangesAbstract" xlink:label="pfsa_ScheduleOfDebtInstrumentsAndTheirChangesAbstract_1298" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentTable" xlink:label="us-gaap_DebtInstrumentTable_1299" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ExtinguishmentOfDebtAmount" xlink:label="us-gaap_ExtinguishmentOfDebtAmount_2250" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RecapitalizationCosts" xlink:label="us-gaap_RecapitalizationCosts_2251" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AccruedStatedInterest" xlink:label="pfsa_AccruedStatedInterest_1329" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentFairValue" xlink:label="us-gaap_DebtInstrumentFairValue_1330" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis_1300" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DebtAccountingBasis" xlink:label="pfsa_DebtAccountingBasis_1318" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="us-gaap_DebtInstrumentInterestRateStatedPercentage_1319" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_1301_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1308_default" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_1301" xlink:to="srt_MaximumMember_1303" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1308" xlink:to="pfsa_TaslyConvertibleDebtRelatedPartyMember_1309" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1308" xlink:to="pfsa_ConvertiblePromissoryNoteRelatedPartyMember_1310" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1308" xlink:to="pfsa_SeniorNoteMember_1311" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1308" xlink:to="pfsa_PromissoryNotesMember_1312" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1308" xlink:to="pfsa_PPPLoanMember_1313" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1308" xlink:to="pfsa_DOInsuranceFinancingMember_1314" />
    <link:definitionArc xlink:type="arc" order="19.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1308" xlink:to="pfsa_JuniorConvertibleNotesMember_1315" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1308" xlink:to="pfsa_LoansPayablesInstrumentMember_2140" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis_1300" xlink:to="srt_RangeMember_1301" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_1301" xlink:to="srt_MinimumMember_1302" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="pfsa_ConversionOfDebtToEquity_1324" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="pfsa_StatedInterest_1325" />
    <link:definitionArc xlink:type="arc" order="19.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="us-gaap_AmortizationOfFinancingCostsAndDiscounts_1326" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="us-gaap_FairValueAdjustmentOfWarrants_1327" />
    <link:definitionArc xlink:type="arc" order="26.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="pfsa_DebtConversionPricesPerShare_1320" />
    <link:definitionArc xlink:type="arc" order="27.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="us-gaap_DebtInstrumentMaturityDate_1321" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="pfsa_DebtRepayments_1322" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="us-gaap_ProceedsFromIssuanceOfDebt_1323" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable_1299" xlink:to="us-gaap_DebtInstrumentAxis_1307" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis_1307" xlink:to="us-gaap_DebtInstrumentNameDomain_1308" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="pfsa_DebtForgiveness_2134" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="us-gaap_DebtInstrumentTable_1299" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="us-gaap_ExtinguishmentOfDebtAmount_2250" />
    <link:definitionArc xlink:type="arc" order="22.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="us-gaap_RecapitalizationCosts_2251" />
    <link:definitionArc xlink:type="arc" order="21.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="pfsa_AccruedStatedInterest_1329" />
    <link:definitionArc xlink:type="arc" order="23.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="us-gaap_DebtInstrumentFairValue_1330" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable_1299" xlink:to="srt_RangeAxis_1300" />
    <link:definitionArc xlink:type="arc" order="24.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="pfsa_DebtAccountingBasis_1318" />
    <link:definitionArc xlink:type="arc" order="25.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems_1316" xlink:to="us-gaap_DebtInstrumentInterestRateStatedPercentage_1319" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis_1300" xlink:to="srt_RangeMember_1301_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis_1307" xlink:to="us-gaap_DebtInstrumentNameDomain_1308_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingSegmentsMember" xlink:label="us-gaap_OperatingSegmentsMember_1430" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss_1449" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TransactionCosts" xlink:label="pfsa_TransactionCosts_1434" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentReportingInformationLineItems" xlink:label="us-gaap_SegmentReportingInformationLineItems_1431" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingExpensesAbstract" xlink:label="us-gaap_OperatingExpensesAbstract_1432" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensation" xlink:label="us-gaap_ShareBasedCompensation_1433" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_UnrealizedGainLossOnDerivatives" xlink:label="us-gaap_UnrealizedGainLossOnDerivatives_1441" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAdjustmentOfWarrants" xlink:label="us-gaap_FairValueAdjustmentOfWarrants_1442" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueOptionChangesInFairValueGainLoss1" xlink:label="us-gaap_FairValueOptionChangesInFairValueGainLoss1_1443" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfOtherFinancialInformationRegardingTheCompanySOperatingSegmentAbstract" xlink:label="pfsa_ScheduleOfOtherFinancialInformationRegardingTheCompanySOperatingSegmentAbstract_1427" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentDetailsTable" xlink:label="pfsa_SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentDetailsTable_1428" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsAxis" xlink:label="srt_ConsolidationItemsAxis_1429" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestExpenseNonoperating" xlink:label="us-gaap_InterestExpenseNonoperating_1444" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GainsLossesOnExtinguishmentOfDebt" xlink:label="us-gaap_GainsLossesOnExtinguishmentOfDebt_1445" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FinancingInterestExpense" xlink:label="us-gaap_FinancingInterestExpense_1446" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsDomain" xlink:label="srt_ConsolidationItemsDomain_2003" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NonoperatingIncomeExpenseAbstract" xlink:label="us-gaap_NonoperatingIncomeExpenseAbstract_2306" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LossOnChangeInFairValueOfDigitalAssets" xlink:label="pfsa_LossOnChangeInFairValueOfDigitalAssets_2307" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ContractResearchOrganizationAndRegulatoryCosts" xlink:label="pfsa_ContractResearchOrganizationAndRegulatoryCosts_2314" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LossOnExtinguishmentOfRelatedPartyDebt" xlink:label="pfsa_LossOnExtinguishmentOfRelatedPartyDebt_2323" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AdministrativePersonnelCompensationCostsIncludingStockbasedCompensation" xlink:label="pfsa_AdministrativePersonnelCompensationCostsIncludingStockbasedCompensation_1435" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_RentAndOfficeCosts" xlink:label="pfsa_RentAndOfficeCosts_1436" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LegalAndAccountingCosts" xlink:label="pfsa_LegalAndAccountingCosts_1437" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherExpenses" xlink:label="us-gaap_OtherExpenses_1438" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingExpenses" xlink:label="us-gaap_OperatingExpenses_1439" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingIncomeLoss" xlink:label="us-gaap_OperatingIncomeLoss_1440" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherNonoperatingIncomeExpense" xlink:label="us-gaap_OtherNonoperatingIncomeExpense_1447" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NonoperatingIncomeExpense" xlink:label="us-gaap_NonoperatingIncomeExpense_1448" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsDomain" xlink:label="srt_ConsolidationItemsDomain_2003_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ConsolidationItemsDomain_2003" xlink:to="us-gaap_OperatingSegmentsMember_1430" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems_1431" xlink:to="us-gaap_NetIncomeLoss_1449" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract_1432" xlink:to="pfsa_TransactionCosts_1434" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems_1431" xlink:to="us-gaap_OperatingExpensesAbstract_1432" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract_1432" xlink:to="us-gaap_ShareBasedCompensation_1433" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="us-gaap_UnrealizedGainLossOnDerivatives_1441" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="us-gaap_FairValueAdjustmentOfWarrants_1442" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="us-gaap_FairValueOptionChangesInFairValueGainLoss1_1443" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_SegmentReportingInformationLineItems_1431" xlink:to="pfsa_SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentDetailsTable_1428" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_SegmentsScheduleofOtherFinancialInformationRegardingTheCompanysOperatingSegmentDetailsTable_1428" xlink:to="srt_ConsolidationItemsAxis_1429" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="us-gaap_InterestExpenseNonoperating_1444" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="us-gaap_GainsLossesOnExtinguishmentOfDebt_1445" />
    <link:definitionArc xlink:type="arc" order="19.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="us-gaap_FinancingInterestExpense_1446" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ConsolidationItemsAxis_1429" xlink:to="srt_ConsolidationItemsDomain_2003" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems_1431" xlink:to="us-gaap_NonoperatingIncomeExpenseAbstract_2306" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="pfsa_LossOnChangeInFairValueOfDigitalAssets_2307" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract_1432" xlink:to="pfsa_ContractResearchOrganizationAndRegulatoryCosts_2314" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="pfsa_LossOnExtinguishmentOfRelatedPartyDebt_2323" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract_1432" xlink:to="pfsa_AdministrativePersonnelCompensationCostsIncludingStockbasedCompensation_1435" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract_1432" xlink:to="pfsa_RentAndOfficeCosts_1436" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract_1432" xlink:to="pfsa_LegalAndAccountingCosts_1437" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract_1432" xlink:to="us-gaap_OtherExpenses_1438" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems_1431" xlink:to="us-gaap_OperatingExpenses_1439" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems_1431" xlink:to="us-gaap_OperatingIncomeLoss_1440" />
    <link:definitionArc xlink:type="arc" order="20.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="us-gaap_OtherNonoperatingIncomeExpense_1447" />
    <link:definitionArc xlink:type="arc" order="21.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract_2306" xlink:to="us-gaap_NonoperatingIncomeExpense_1448" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ConsolidationItemsAxis_1429" xlink:to="srt_ConsolidationItemsDomain_2003_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/NetLossPerShareAttributabletoCommonStockholdersDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_EarnoutSharesMember" xlink:label="pfsa_EarnoutSharesMember_1649" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AscentPIPENotesMember" xlink:label="pfsa_AscentPIPENotesMember_1652" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TaslyConvertibleNotesMember" xlink:label="pfsa_TaslyConvertibleNotesMember_1655" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TradingDays" xlink:label="pfsa_TradingDays_2223" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis_1650" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1651" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionAxis" xlink:label="us-gaap_RelatedPartyTransactionAxis_1653" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain_1654" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ContractualFloorPrice" xlink:label="pfsa_ContractualFloorPrice_2255" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_UndistributedEarningsLossAllocatedToParticipatingSecuritiesBasic" xlink:label="us-gaap_UndistributedEarningsLossAllocatedToParticipatingSecuritiesBasic_2292" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EarningsPerShareAbstract" xlink:label="us-gaap_EarningsPerShareAbstract_1645" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_NetLossPerShareAttributabletoCommonStockholdersDetailsTable" xlink:label="pfsa_NetLossPerShareAttributabletoCommonStockholdersDetailsTable_1646" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_1647" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1648" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EarningsPerShareBasicLineItems" xlink:label="us-gaap_EarningsPerShareBasicLineItems_1657" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConversionOfStockSharesIssued1" xlink:label="us-gaap_ConversionOfStockSharesIssued1_1658" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockConvertibleConversionPriceIncrease" xlink:label="us-gaap_CommonStockConvertibleConversionPriceIncrease_1659" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PercentageOfClosingSharePrice" xlink:label="pfsa_PercentageOfClosingSharePrice_1660" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues_1662" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_1651_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain_1654_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1648_default" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1648" xlink:to="pfsa_EarnoutSharesMember_1649" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain_1651" xlink:to="pfsa_AscentPIPENotesMember_1652" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain_1654" xlink:to="pfsa_TaslyConvertibleNotesMember_1655" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareBasicLineItems_1657" xlink:to="pfsa_TradingDays_2223" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_NetLossPerShareAttributabletoCommonStockholdersDetailsTable_1646" xlink:to="us-gaap_DebtInstrumentAxis_1650" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis_1650" xlink:to="us-gaap_DebtInstrumentNameDomain_1651" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_NetLossPerShareAttributabletoCommonStockholdersDetailsTable_1646" xlink:to="us-gaap_RelatedPartyTransactionAxis_1653" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionAxis_1653" xlink:to="us-gaap_RelatedPartyTransactionDomain_1654" />
    <link:definitionArc xlink:type="arc" order="295.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareBasicLineItems_1657" xlink:to="pfsa_ContractualFloorPrice_2255" />
    <link:definitionArc xlink:type="arc" order="328.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareBasicLineItems_1657" xlink:to="us-gaap_UndistributedEarningsLossAllocatedToParticipatingSecuritiesBasic_2292" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_EarningsPerShareBasicLineItems_1657" xlink:to="pfsa_NetLossPerShareAttributabletoCommonStockholdersDetailsTable_1646" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_NetLossPerShareAttributabletoCommonStockholdersDetailsTable_1646" xlink:to="us-gaap_StatementClassOfStockAxis_1647" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_1647" xlink:to="us-gaap_ClassOfStockDomain_1648" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareBasicLineItems_1657" xlink:to="us-gaap_ConversionOfStockSharesIssued1_1658" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareBasicLineItems_1657" xlink:to="us-gaap_CommonStockConvertibleConversionPriceIncrease_1659" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareBasicLineItems_1657" xlink:to="pfsa_PercentageOfClosingSharePrice_1660" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareBasicLineItems_1657" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues_1662" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis_1650" xlink:to="us-gaap_DebtInstrumentNameDomain_1651_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionAxis_1653" xlink:to="us-gaap_RelatedPartyTransactionDomain_1654_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_1647" xlink:to="us-gaap_ClassOfStockDomain_1648_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/StockholdersEquityDeficitDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2430" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember_2431" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember_2432" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FinancialAdvisorsAndVendorsMember" xlink:label="pfsa_FinancialAdvisorsAndVendorsMember_2451" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DesignatedSeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_DesignatedSeriesANon-VotingConvertiblePreferredStockMember_2391" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_UndesignatedSeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_UndesignatedSeriesANon-VotingConvertiblePreferredStockMember_2392" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2410" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_EarnoutSharesMember" xlink:label="pfsa_EarnoutSharesMember_1607" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_GainLossOnSettlementLiabilities" xlink:label="pfsa_GainLossOnSettlementLiabilities_2515" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GainsLossesOnExtinguishmentOfDebt" xlink:label="us-gaap_GainsLossesOnExtinguishmentOfDebt_2516" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockDividendsPerShareDeclared" xlink:label="us-gaap_CommonStockDividendsPerShareDeclared_2425" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_BeneficialOwnershipLimitationPercentage" xlink:label="pfsa_BeneficialOwnershipLimitationPercentage_2426" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleDebtCurrent" xlink:label="us-gaap_ConvertibleDebtCurrent_2427" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis_2429" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SubjectToPossibleRedemption" xlink:label="pfsa_SubjectToPossibleRedemption_2433" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConversionOfStockSharesConverted1" xlink:label="us-gaap_ConversionOfStockSharesConverted1_2435" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesAverageAmountOutstanding" xlink:label="us-gaap_LiabilitiesAverageAmountOutstanding_2436" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_2449" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_2450" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="us-gaap_PreferredStockSharesAuthorized_2337" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="us-gaap_PreferredStockParOrStatedValuePerShare_2338" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction" xlink:label="us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_2397" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense_2405" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense_2406" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PaymentsOfFinancingCosts" xlink:label="us-gaap_PaymentsOfFinancingCosts_2408" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenuesNetOfInterestExpense" xlink:label="us-gaap_RevenuesNetOfInterestExpense_2409" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesReverseStockSplits" xlink:label="us-gaap_StockIssuedDuringPeriodSharesReverseStockSplits_2421" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueOfPreferredStock" xlink:label="pfsa_FairValueOfPreferredStock_2422" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccruedBonusesCurrentAndNoncurrent" xlink:label="us-gaap_AccruedBonusesCurrentAndNoncurrent_2424" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityNoteAbstract" xlink:label="us-gaap_StockholdersEquityNoteAbstract_1603" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_StockholdersDeficitDetailsTable" xlink:label="pfsa_StockholdersDeficitDetailsTable_1604" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_1605" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1606" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockLineItems" xlink:label="us-gaap_ClassOfStockLineItems_1608" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesIssued" xlink:label="us-gaap_PreferredStockSharesIssued_1609" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="us-gaap_PreferredStockSharesOutstanding_1610" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockVotingRights" xlink:label="us-gaap_CommonStockVotingRights_1611" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DividendsCommonStockCash" xlink:label="us-gaap_DividendsCommonStockCash_1612" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues_1613" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2430_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_2450_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1606_default" />
    <link:definitionArc xlink:type="arc" order="435.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis_2429" xlink:to="srt_RangeMember_2430" />
    <link:definitionArc xlink:type="arc" order="436.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2430" xlink:to="srt_MaximumMember_2431" />
    <link:definitionArc xlink:type="arc" order="437.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2430" xlink:to="srt_MinimumMember_2432" />
    <link:definitionArc xlink:type="arc" order="450.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain_2450" xlink:to="pfsa_FinancialAdvisorsAndVendorsMember_2451" />
    <link:definitionArc xlink:type="arc" order="400.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1606" xlink:to="pfsa_DesignatedSeriesANon-VotingConvertiblePreferredStockMember_2391" />
    <link:definitionArc xlink:type="arc" order="401.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1606" xlink:to="pfsa_UndesignatedSeriesANon-VotingConvertiblePreferredStockMember_2392" />
    <link:definitionArc xlink:type="arc" order="417.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1606" xlink:to="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2410" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1606" xlink:to="pfsa_EarnoutSharesMember_1607" />
    <link:definitionArc xlink:type="arc" order="501.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="pfsa_GainLossOnSettlementLiabilities_2515" />
    <link:definitionArc xlink:type="arc" order="502.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_GainsLossesOnExtinguishmentOfDebt_2516" />
    <link:definitionArc xlink:type="arc" order="430.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_CommonStockDividendsPerShareDeclared_2425" />
    <link:definitionArc xlink:type="arc" order="431.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="pfsa_BeneficialOwnershipLimitationPercentage_2426" />
    <link:definitionArc xlink:type="arc" order="432.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_ConvertibleDebtCurrent_2427" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_StockholdersDeficitDetailsTable_1604" xlink:to="srt_RangeAxis_2429" />
    <link:definitionArc xlink:type="arc" order="438.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="pfsa_SubjectToPossibleRedemption_2433" />
    <link:definitionArc xlink:type="arc" order="440.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_ConversionOfStockSharesConverted1_2435" />
    <link:definitionArc xlink:type="arc" order="441.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_LiabilitiesAverageAmountOutstanding_2436" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_StockholdersDeficitDetailsTable_1604" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_2449" />
    <link:definitionArc xlink:type="arc" order="449.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_2449" xlink:to="us-gaap_RelatedPartyDomain_2450" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_PreferredStockSharesAuthorized_2337" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_PreferredStockParOrStatedValuePerShare_2338" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_2397" />
    <link:definitionArc xlink:type="arc" order="20.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_ResearchAndDevelopmentExpense_2405" />
    <link:definitionArc xlink:type="arc" order="19.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_GeneralAndAdministrativeExpense_2406" />
    <link:definitionArc xlink:type="arc" order="21.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_PaymentsOfFinancingCosts_2408" />
    <link:definitionArc xlink:type="arc" order="22.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_RevenuesNetOfInterestExpense_2409" />
    <link:definitionArc xlink:type="arc" order="426.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_StockIssuedDuringPeriodSharesReverseStockSplits_2421" />
    <link:definitionArc xlink:type="arc" order="427.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="pfsa_FairValueOfPreferredStock_2422" />
    <link:definitionArc xlink:type="arc" order="429.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_AccruedBonusesCurrentAndNoncurrent_2424" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="pfsa_StockholdersDeficitDetailsTable_1604" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_StockholdersDeficitDetailsTable_1604" xlink:to="us-gaap_StatementClassOfStockAxis_1605" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_1605" xlink:to="us-gaap_ClassOfStockDomain_1606" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_PreferredStockSharesIssued_1609" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_PreferredStockSharesOutstanding_1610" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_CommonStockVotingRights_1611" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_DividendsCommonStockCash_1612" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_1608" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues_1613" />
    <link:definitionArc xlink:type="arc" order="435.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis_2429" xlink:to="srt_RangeMember_2430_default" />
    <link:definitionArc xlink:type="arc" order="449.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_2449" xlink:to="us-gaap_RelatedPartyDomain_2450_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_1605" xlink:to="us-gaap_ClassOfStockDomain_1606_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/CommonStockWarrantsScheduleofFollowingKeyInputsasAffectedbytheReverseStockSplitsDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2070" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember_2071" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember_2072" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfFollowingKeyInputsAsAffectedByTheReverseStockSplitsLineItems" xlink:label="pfsa_ScheduleOfFollowingKeyInputsAsAffectedByTheReverseStockSplitsLineItems_2187" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ShareBasedPaymentArrangementDisclosureAbstract" xlink:label="pfsa_ShareBasedPaymentArrangementDisclosureAbstract_2060" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_2061" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_2062" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExercisePrice_2065" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice_2066" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_2067" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_2068" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis_2069" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2070_default" />
    <link:definitionArc xlink:type="arc" order="162.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis_2069" xlink:to="srt_RangeMember_2070" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2070" xlink:to="srt_MaximumMember_2071" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2070" xlink:to="srt_MinimumMember_2072" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="pfsa_ScheduleOfFollowingKeyInputsAsAffectedByTheReverseStockSplitsLineItems_2187" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_2061" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="pfsa_ScheduleOfFollowingKeyInputsAsAffectedByTheReverseStockSplitsLineItems_2187" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_2062" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="pfsa_ScheduleOfFollowingKeyInputsAsAffectedByTheReverseStockSplitsLineItems_2187" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExercisePrice_2065" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="pfsa_ScheduleOfFollowingKeyInputsAsAffectedByTheReverseStockSplitsLineItems_2187" xlink:to="us-gaap_SharePrice_2066" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="pfsa_ScheduleOfFollowingKeyInputsAsAffectedByTheReverseStockSplitsLineItems_2187" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_2067" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="pfsa_ScheduleOfFollowingKeyInputsAsAffectedByTheReverseStockSplitsLineItems_2187" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_2068" />
    <link:definitionArc xlink:type="arc" order="161.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_2068" xlink:to="srt_RangeAxis_2069" />
    <link:definitionArc xlink:type="arc" order="162.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis_2069" xlink:to="srt_RangeMember_2070_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/RelatedPartyTransactionsDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AdministrativeServiceFeeMember" xlink:label="pfsa_AdministrativeServiceFeeMember_2125" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyMember" xlink:label="us-gaap_RelatedPartyMember_1640" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_StatementOfIncomeLocationAxis" xlink:label="pfsa_StatementOfIncomeLocationAxis_2123" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_StatementOfIncomeLocationDomain" xlink:label="pfsa_StatementOfIncomeLocationDomain_2124" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionLineItems" xlink:label="us-gaap_RelatedPartyTransactionLineItems_1641" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestExpense" xlink:label="us-gaap_InterestExpense_1642" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionAmountsOfTransaction" xlink:label="us-gaap_RelatedPartyTransactionAmountsOfTransaction_1643" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GainsLossesOnExtinguishmentOfDebt" xlink:label="us-gaap_GainsLossesOnExtinguishmentOfDebt_2364" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_DebtExtinguishmentOnLoss" xlink:label="pfsa_DebtExtinguishmentOnLoss_2512" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionsAbstract" xlink:label="us-gaap_RelatedPartyTransactionsAbstract_1635" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:label="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_1636" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1638" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_1639" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_StatementOfIncomeLocationDomain" xlink:label="pfsa_StatementOfIncomeLocationDomain_2124_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_1639_default" />
    <link:definitionArc xlink:type="arc" order="209.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="pfsa_StatementOfIncomeLocationDomain_2124" xlink:to="pfsa_AdministrativeServiceFeeMember_2125" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain_1639" xlink:to="us-gaap_RelatedPartyMember_1640" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_1636" xlink:to="pfsa_StatementOfIncomeLocationAxis_2123" />
    <link:definitionArc xlink:type="arc" order="208.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="pfsa_StatementOfIncomeLocationAxis_2123" xlink:to="pfsa_StatementOfIncomeLocationDomain_2124" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionLineItems_1641" xlink:to="us-gaap_InterestExpense_1642" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionLineItems_1641" xlink:to="us-gaap_RelatedPartyTransactionAmountsOfTransaction_1643" />
    <link:definitionArc xlink:type="arc" order="378.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionLineItems_1641" xlink:to="us-gaap_GainsLossesOnExtinguishmentOfDebt_2364" />
    <link:definitionArc xlink:type="arc" order="498.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionLineItems_1641" xlink:to="pfsa_DebtExtinguishmentOnLoss_2512" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_RelatedPartyTransactionLineItems_1641" xlink:to="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_1636" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_1636" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1638" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1638" xlink:to="us-gaap_RelatedPartyDomain_1639" />
    <link:definitionArc xlink:type="arc" order="208.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="pfsa_StatementOfIncomeLocationAxis_2123" xlink:to="pfsa_StatementOfIncomeLocationDomain_2124_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1638" xlink:to="us-gaap_RelatedPartyDomain_1639_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/StockholdersEquityDeficitScheduleofReservedSharesofCommonStockforFutureIssuanceDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_OutstandingOptionsUnderTwoThousandTenPlanMember" xlink:label="pfsa_OutstandingOptionsUnderTwoThousandTenPlanMember_1353" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfOptionsUnderTheTwoThousandTenPlanMember" xlink:label="pfsa_IssuanceOfOptionsUnderTheTwoThousandTenPlanMember_1354" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_OutstandingCommonStockWarrantsMember" xlink:label="pfsa_OutstandingCommonStockWarrantsMember_1355" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfEarnoutSharesMember" xlink:label="pfsa_IssuanceOfEarnoutSharesMember_1356" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConversionOfSeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_ConversionOfSeriesANon-VotingConvertiblePreferredStockMember_2300" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfReservedSharesOfCommonStockForFutureIssuanceAbstract" xlink:label="pfsa_ScheduleOfReservedSharesOfCommonStockForFutureIssuanceAbstract_1349" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConversionOfStockTable" xlink:label="us-gaap_ConversionOfStockTable_1350" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_1351" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1352" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConversionOfStockLineItems" xlink:label="us-gaap_ConversionOfStockLineItems_1357" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:label="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_1358" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1352_default" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1352" xlink:to="pfsa_OutstandingOptionsUnderTwoThousandTenPlanMember_1353" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1352" xlink:to="pfsa_IssuanceOfOptionsUnderTheTwoThousandTenPlanMember_1354" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1352" xlink:to="pfsa_OutstandingCommonStockWarrantsMember_1355" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1352" xlink:to="pfsa_IssuanceOfEarnoutSharesMember_1356" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1352" xlink:to="pfsa_ConversionOfSeriesANon-VotingConvertiblePreferredStockMember_2300" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ConversionOfStockLineItems_1357" xlink:to="us-gaap_ConversionOfStockTable_1350" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ConversionOfStockTable_1350" xlink:to="us-gaap_StatementClassOfStockAxis_1351" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_1351" xlink:to="us-gaap_ClassOfStockDomain_1352" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockLineItems_1357" xlink:to="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_1358" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_1351" xlink:to="us-gaap_ClassOfStockDomain_1352_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/CommitmentsandContingenciesDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2441" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1585" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_NewLeaseAgreementMember" xlink:label="pfsa_NewLeaseAgreementMember_1586" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_OperatingLeaseObligationsMember" xlink:label="pfsa_OperatingLeaseObligationsMember_2052" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_OptionAgreementMember" xlink:label="pfsa_OptionAgreementMember_2270" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingenciesLineItems" xlink:label="us-gaap_LossContingenciesLineItems_1590" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingLeaseCost" xlink:label="us-gaap_OperatingLeaseCost_1591" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_2258" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_2260" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ManagementBonuses" xlink:label="pfsa_ManagementBonuses_2263" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PayableInCash" xlink:label="pfsa_PayableInCash_2265" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PercentageOfNetRevenues" xlink:label="pfsa_PercentageOfNetRevenues_2150" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromLegalSettlements" xlink:label="us-gaap_ProceedsFromLegalSettlements_1600" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_UnpaidWages" xlink:label="pfsa_UnpaidWages_1601" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AmountsPayable" xlink:label="pfsa_AmountsPayable_2511" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PayableInShares" xlink:label="pfsa_PayableInShares_2267" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommitmentsAndContingenciesDisclosureAbstract" xlink:label="us-gaap_CommitmentsAndContingenciesDisclosureAbstract_1582" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_CommitmentsandContingenciesDetailsTable" xlink:label="pfsa_CommitmentsandContingenciesDetailsTable_1583" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis_1584" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubordinatedBorrowingInterestRate" xlink:label="us-gaap_SubordinatedBorrowingInterestRate_1592" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingLeaseExpense" xlink:label="us-gaap_OperatingLeaseExpense_1593" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubleaseIncome" xlink:label="us-gaap_SubleaseIncome_1594" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermLeaseCost" xlink:label="us-gaap_ShortTermLeaseCost_1596" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingLeaseLiability" xlink:label="us-gaap_OperatingLeaseLiability_1598" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1585_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_2260_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_2260" xlink:to="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2441" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis_1584" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1585" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1585" xlink:to="pfsa_NewLeaseAgreementMember_1586" />
    <link:definitionArc xlink:type="arc" order="145.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1585" xlink:to="pfsa_OperatingLeaseObligationsMember_2052" />
    <link:definitionArc xlink:type="arc" order="146.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1585" xlink:to="pfsa_OptionAgreementMember_2270" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="us-gaap_OperatingLeaseCost_1591" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_CommitmentsandContingenciesDetailsTable_1583" xlink:to="us-gaap_StatementClassOfStockAxis_2258" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_2258" xlink:to="us-gaap_ClassOfStockDomain_2260" />
    <link:definitionArc xlink:type="arc" order="19.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="pfsa_ManagementBonuses_2263" />
    <link:definitionArc xlink:type="arc" order="20.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="pfsa_PayableInCash_2265" />
    <link:definitionArc xlink:type="arc" order="26.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="pfsa_PercentageOfNetRevenues_2150" />
    <link:definitionArc xlink:type="arc" order="23.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="us-gaap_ProceedsFromLegalSettlements_1600" />
    <link:definitionArc xlink:type="arc" order="24.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="pfsa_UnpaidWages_1601" />
    <link:definitionArc xlink:type="arc" order="497.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="pfsa_AmountsPayable_2511" />
    <link:definitionArc xlink:type="arc" order="21.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="pfsa_PayableInShares_2267" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="pfsa_CommitmentsandContingenciesDetailsTable_1583" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="pfsa_CommitmentsandContingenciesDetailsTable_1583" xlink:to="us-gaap_TypeOfArrangementAxis_1584" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="us-gaap_SubordinatedBorrowingInterestRate_1592" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="us-gaap_OperatingLeaseExpense_1593" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="us-gaap_SubleaseIncome_1594" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="us-gaap_ShortTermLeaseCost_1596" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LossContingenciesLineItems_1590" xlink:to="us-gaap_OperatingLeaseLiability_1598" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis_1584" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_1585_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_2258" xlink:to="us-gaap_ClassOfStockDomain_2260_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/ProposedAcquisitionofG3Details" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2442" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2347" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_OptionAgreementMember" xlink:label="pfsa_OptionAgreementMember_2348" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockAndCommonStockInConnectionWithG3CallOptionIssuanceInShares" xlink:label="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockAndCommonStockInConnectionWithG3CallOptionIssuanceInShares_2339" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_2343" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_2344" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis_2346" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AdditionalSharesOfSeriesANon-VotingConvertiblePreferredStock" xlink:label="pfsa_AdditionalSharesOfSeriesANon-VotingConvertiblePreferredStock_2349" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_GrossProceeds" xlink:label="pfsa_GrossProceeds_2350" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PayableInCash" xlink:label="pfsa_PayableInCash_2352" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction" xlink:label="us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_2354" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_CallOptionExpirationDate" xlink:label="pfsa_CallOptionExpirationDate_2356" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueOfInitialConsideration" xlink:label="pfsa_FairValueOfInitialConsideration_2357" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LegalAndOtherProfessionalFees" xlink:label="pfsa_LegalAndOtherProfessionalFees_2358" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SellerLiquidatedDamagesPercentage" xlink:label="pfsa_SellerLiquidatedDamagesPercentage_2359" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_InitialConsiderationFilingDeadline" xlink:label="pfsa_InitialConsiderationFilingDeadline_2362" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TransactionCosts" xlink:label="pfsa_TransactionCosts_2377" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TransactionFees" xlink:label="pfsa_TransactionFees_2381" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccruedBonusesCurrent" xlink:label="us-gaap_AccruedBonusesCurrent_2384" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetAcquisitionAbstract" xlink:label="us-gaap_AssetAcquisitionAbstract_2312" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetAcquisitionTable" xlink:label="us-gaap_AssetAcquisitionTable_2313" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetAcquisitionLineItems" xlink:label="us-gaap_AssetAcquisitionLineItems_2325" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2347_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_2344_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_2344" xlink:to="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2442" />
    <link:definitionArc xlink:type="arc" order="363.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis_2346" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2347" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2347" xlink:to="pfsa_OptionAgreementMember_2348" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockAndCommonStockInConnectionWithG3CallOptionIssuanceInShares_2339" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_AssetAcquisitionTable_2313" xlink:to="us-gaap_StatementClassOfStockAxis_2343" />
    <link:definitionArc xlink:type="arc" order="362.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_2343" xlink:to="us-gaap_ClassOfStockDomain_2344" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_AssetAcquisitionTable_2313" xlink:to="us-gaap_TypeOfArrangementAxis_2346" />
    <link:definitionArc xlink:type="arc" order="364.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_AdditionalSharesOfSeriesANon-VotingConvertiblePreferredStock_2349" />
    <link:definitionArc xlink:type="arc" order="365.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_GrossProceeds_2350" />
    <link:definitionArc xlink:type="arc" order="367.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_PayableInCash_2352" />
    <link:definitionArc xlink:type="arc" order="369.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_2354" />
    <link:definitionArc xlink:type="arc" order="370.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_CallOptionExpirationDate_2356" />
    <link:definitionArc xlink:type="arc" order="371.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_FairValueOfInitialConsideration_2357" />
    <link:definitionArc xlink:type="arc" order="372.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_LegalAndOtherProfessionalFees_2358" />
    <link:definitionArc xlink:type="arc" order="373.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_SellerLiquidatedDamagesPercentage_2359" />
    <link:definitionArc xlink:type="arc" order="376.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_InitialConsiderationFilingDeadline_2362" />
    <link:definitionArc xlink:type="arc" order="377.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_TransactionCosts_2377" />
    <link:definitionArc xlink:type="arc" order="390.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="pfsa_TransactionFees_2381" />
    <link:definitionArc xlink:type="arc" order="393.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="us-gaap_AccruedBonusesCurrent_2384" />
    <link:definitionArc xlink:type="arc" order="338.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_AssetAcquisitionLineItems_2325" xlink:to="us-gaap_AssetAcquisitionTable_2313" />
    <link:definitionArc xlink:type="arc" order="363.00000" priority="1" xbrldt:closed="true" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis_2346" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2347_default" />
    <link:definitionArc xlink:type="arc" order="362.00000" priority="1" xbrldt:closed="true" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_2343" xlink:to="us-gaap_ClassOfStockDomain_2344_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/RelatedPartyTransactionsScheduleofRelatedPartyTransactionsDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TaslyMember" xlink:label="pfsa_TaslyMember_1395" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_NVACSponsorMember" xlink:label="pfsa_NVACSponsorMember_1396" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_TheFoundersMember" xlink:label="pfsa_TheFoundersMember_1397" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_NVACMember" xlink:label="pfsa_NVACMember_1398" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionLineItems" xlink:label="us-gaap_RelatedPartyTransactionLineItems_1399" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NatureOfCommonOwnershipOrManagementControlRelationships" xlink:label="us-gaap_NatureOfCommonOwnershipOrManagementControlRelationships_1400" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionDescriptionOfTransaction" xlink:label="us-gaap_RelatedPartyTransactionDescriptionOfTransaction_1401" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionAmountsOfTransaction" xlink:label="us-gaap_RelatedPartyTransactionAmountsOfTransaction_1402" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfRelatedPartyTransactionsAbstract" xlink:label="pfsa_ScheduleOfRelatedPartyTransactionsAbstract_1392" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:label="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_1393" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1394" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_2002" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_2002_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain_2002" xlink:to="pfsa_TaslyMember_1395" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain_2002" xlink:to="pfsa_NVACSponsorMember_1396" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain_2002" xlink:to="pfsa_TheFoundersMember_1397" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain_2002" xlink:to="pfsa_NVACMember_1398" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionLineItems_1399" xlink:to="us-gaap_NatureOfCommonOwnershipOrManagementControlRelationships_1400" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionLineItems_1399" xlink:to="us-gaap_RelatedPartyTransactionDescriptionOfTransaction_1401" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionLineItems_1399" xlink:to="us-gaap_RelatedPartyTransactionAmountsOfTransaction_1402" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_RelatedPartyTransactionLineItems_1399" xlink:to="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_1393" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_1393" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1394" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1394" xlink:to="us-gaap_RelatedPartyDomain_2002" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1394" xlink:to="us-gaap_RelatedPartyDomain_2002_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/CommonStockWarrantsScheduleofClassesofWarrantsDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PublicWarrantsMember" xlink:label="pfsa_PublicWarrantsMember_1364" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_PrivatePlacementWarrantsMember" xlink:label="pfsa_PrivatePlacementWarrantsMember_1365" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_RepresentativesWarrantsMember" xlink:label="pfsa_RepresentativesWarrantsMember_1366" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_HCWWarrantsMember" xlink:label="pfsa_HCWWarrantsMember_1367" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AscentWarrantsMember" xlink:label="pfsa_AscentWarrantsMember_2055" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_1369" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRighstDateFromWhichWarrantsOrRightsExercisable" xlink:label="us-gaap_ClassOfWarrantOrRighstDateFromWhichWarrantsOrRightsExercisable_1370" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:label="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_1371" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfClassesOfWarrantsAbstract" xlink:label="pfsa_ScheduleOfClassesOfWarrantsAbstract_1360" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightTable" xlink:label="us-gaap_ClassOfWarrantOrRightTable_1361" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis_1362" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_1363" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightLineItems" xlink:label="us-gaap_ClassOfWarrantOrRightLineItems_1368" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_1363_default" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1363" xlink:to="pfsa_PublicWarrantsMember_1364" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1363" xlink:to="pfsa_PrivatePlacementWarrantsMember_1365" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1363" xlink:to="pfsa_RepresentativesWarrantsMember_1366" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1363" xlink:to="pfsa_HCWWarrantsMember_1367" />
    <link:definitionArc xlink:type="arc" order="148.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain_1363" xlink:to="pfsa_AscentWarrantsMember_2055" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightLineItems_1368" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_1369" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightLineItems_1368" xlink:to="us-gaap_ClassOfWarrantOrRighstDateFromWhichWarrantsOrRightsExercisable_1370" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightLineItems_1368" xlink:to="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_1371" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfWarrantOrRightLineItems_1368" xlink:to="us-gaap_ClassOfWarrantOrRightTable_1361" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ClassOfWarrantOrRightTable_1361" xlink:to="us-gaap_ClassOfWarrantOrRightAxis_1362" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1362" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_1363" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis_1362" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_1363_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/StockholdersEquityDeficitScheduleofConvertiblePreferredStockDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2375" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember_2378" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember_2379" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputOptionVolatilityMember" xlink:label="us-gaap_MeasurementInputOptionVolatilityMember_2320" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:label="us-gaap_MeasurementInputRiskFreeInterestRateMember_2321" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputExpectedTermMember" xlink:label="us-gaap_MeasurementInputExpectedTermMember_2322" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputDiscountForLackOfMarketabilityMember" xlink:label="us-gaap_MeasurementInputDiscountForLackOfMarketabilityMember_2326" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputConversionPriceMember" xlink:label="us-gaap_MeasurementInputConversionPriceMember_2327" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ProbabilityOfObtainingRequiredFinancingMember" xlink:label="pfsa_ProbabilityOfObtainingRequiredFinancingMember_2328" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueOfCommonStocksMember" xlink:label="pfsa_FairValueOfCommonStocksMember_2329" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockLineItems" xlink:label="us-gaap_ClassOfStockLineItems_2373" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis_2374" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityAbstract" xlink:label="us-gaap_EquityAbstract_2315" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:label="us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_2316" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="us-gaap_MeasurementInputTypeAxis_2318" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain_2319" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentMeasurementInput" xlink:label="us-gaap_DebtInstrumentMeasurementInput_2402" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2375_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain_2319_default" />
    <link:definitionArc xlink:type="arc" order="385.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis_2374" xlink:to="srt_RangeMember_2375" />
    <link:definitionArc xlink:type="arc" order="387.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2375" xlink:to="srt_MinimumMember_2378" />
    <link:definitionArc xlink:type="arc" order="388.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2375" xlink:to="srt_MaximumMember_2379" />
    <link:definitionArc xlink:type="arc" order="343.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2319" xlink:to="us-gaap_MeasurementInputOptionVolatilityMember_2320" />
    <link:definitionArc xlink:type="arc" order="344.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2319" xlink:to="us-gaap_MeasurementInputRiskFreeInterestRateMember_2321" />
    <link:definitionArc xlink:type="arc" order="345.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2319" xlink:to="us-gaap_MeasurementInputExpectedTermMember_2322" />
    <link:definitionArc xlink:type="arc" order="349.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2319" xlink:to="us-gaap_MeasurementInputDiscountForLackOfMarketabilityMember_2326" />
    <link:definitionArc xlink:type="arc" order="350.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2319" xlink:to="us-gaap_MeasurementInputConversionPriceMember_2327" />
    <link:definitionArc xlink:type="arc" order="351.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2319" xlink:to="pfsa_ProbabilityOfObtainingRequiredFinancingMember_2328" />
    <link:definitionArc xlink:type="arc" order="352.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2319" xlink:to="pfsa_FairValueOfCommonStocksMember_2329" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_2316" xlink:to="srt_RangeAxis_2374" />
    <link:definitionArc xlink:type="arc" order="340.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfStockLineItems_2373" xlink:to="us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_2316" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_2316" xlink:to="us-gaap_MeasurementInputTypeAxis_2318" />
    <link:definitionArc xlink:type="arc" order="342.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_MeasurementInputTypeAxis_2318" xlink:to="us-gaap_MeasurementInputTypeDomain_2319" />
    <link:definitionArc xlink:type="arc" order="411.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems_2373" xlink:to="us-gaap_DebtInstrumentMeasurementInput_2402" />
    <link:definitionArc xlink:type="arc" order="385.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis_2374" xlink:to="srt_RangeMember_2375_default" />
    <link:definitionArc xlink:type="arc" order="342.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_MeasurementInputTypeAxis_2318" xlink:to="us-gaap_MeasurementInputTypeDomain_2319_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/CondensedConsolidatedStatementsofConvertiblePreferredStockandStockholdersEquityDeficitUnaudited" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockMember" xlink:label="us-gaap_PreferredStockMember_1081" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember_1082" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="us-gaap_AdditionalPaidInCapitalMember_1083" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsMember" xlink:label="us-gaap_RetainedEarningsMember_1084" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SeriesAPreferredStockMember" xlink:label="us-gaap_SeriesAPreferredStockMember_1076" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SeriesBPreferredStockMember" xlink:label="us-gaap_SeriesBPreferredStockMember_1077" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2457" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SeriesCPreferredStockMember" xlink:label="us-gaap_SeriesCPreferredStockMember_1078" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalWarrantIssued" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalWarrantIssued_2008" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_AdjustmentsToAdditionalPaidInCapitalExciseTaxPayableAttributableToRedemptionOfCommonStock" xlink:label="pfsa_AdjustmentsToAdditionalPaidInCapitalExciseTaxPayableAttributableToRedemptionOfCommonStock_2009" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1079" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis_1080" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesOther" xlink:label="us-gaap_StockIssuedDuringPeriodSharesOther_1092" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_1093" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss_1094" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity_1095" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesOutstanding" xlink:label="us-gaap_SharesOutstanding_1096" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfSharesInConnectionWithTheConversionOfTheRelatedPartyConvertiblePromissoryNote" xlink:label="pfsa_IssuanceOfSharesInConnectionWithTheConversionOfTheRelatedPartyConvertiblePromissoryNote_2284" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfSharesInConnectionWithTheConversionOfTheRelatedPartyConvertiblePromissoryNoteInShares" xlink:label="pfsa_IssuanceOfSharesInConnectionWithTheConversionOfTheRelatedPartyConvertiblePromissoryNoteInShares_2285" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfSharesInConnectionWithTheConversionOfThePipeNote" xlink:label="pfsa_IssuanceOfSharesInConnectionWithTheConversionOfThePipeNote_2289" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfSharesInConnectionWithTheConversionOfThePipeNoteInShares" xlink:label="pfsa_IssuanceOfSharesInConnectionWithTheConversionOfThePipeNoteInShares_2291" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_CancellationOfFractionalSharesInConnectionWithAugustReverseStockSplit" xlink:label="pfsa_CancellationOfFractionalSharesInConnectionWithAugustReverseStockSplit_2296" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_CancellationOfFractionalSharesInConnectionWithAugustReverseStockSplitInShares" xlink:label="pfsa_CancellationOfFractionalSharesInConnectionWithAugustReverseStockSplitInShares_2297" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities" xlink:label="us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities_2330" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" xlink:label="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_2331" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockOwnershipPlan" xlink:label="us-gaap_StockIssuedDuringPeriodValueEmployeeStockOwnershipPlan_2332" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockOwnershipPlan" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockOwnershipPlan_2333" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementOfStockholdersEquityAbstract" xlink:label="us-gaap_StatementOfStockholdersEquityAbstract_1073" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable_1074" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_1075" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain_1085" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems_1086" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConversionOfDebtToCommonSharesInConnectionWithTheMerger" xlink:label="pfsa_ConversionOfDebtToCommonSharesInConnectionWithTheMerger_2336" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConversionOfDebtToCommonSharesInConnectionWithTheMergerInShares" xlink:label="pfsa_ConversionOfDebtToCommonSharesInConnectionWithTheMergerInShares_2360" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfCommonStockToNorthviewStockholdersAsAResultOfTheMerger" xlink:label="pfsa_IssuanceOfCommonStockToNorthviewStockholdersAsAResultOfTheMerger_2367" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfCommonStockToNorthvieStockholdersAsAResultOfTheMerger" xlink:label="pfsa_IssuanceOfCommonStockToNorthvieStockholdersAsAResultOfTheMerger_2368" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfInducedCommonSharesToRelatedPartyInConnectionWithTheMerger" xlink:label="pfsa_IssuanceOfInducedCommonSharesToRelatedPartyInConnectionWithTheMerger_2369" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_StockIssuedDuringPeriodValueIssuanceOfWarrantsToFinancialAdvisorInConnectionWithTheMerger" xlink:label="pfsa_StockIssuedDuringPeriodValueIssuanceOfWarrantsToFinancialAdvisorInConnectionWithTheMerger_2458" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_StockIssuedDuringPeriodSharesIssuanceOfInducedCommonSharesToRelatedPartyInConnectionWithTheMerger" xlink:label="pfsa_StockIssuedDuringPeriodSharesIssuanceOfInducedCommonSharesToRelatedPartyInConnectionWithTheMerger_2459" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues_1089" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockAndCommonStockInConnectionWithG3CallOptionIssuanceInShares" xlink:label="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockAndCommonStockInConnectionWithG3CallOptionIssuanceInShares_2277" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues_1090" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueOther" xlink:label="us-gaap_StockIssuedDuringPeriodValueOther_1091" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockForSettlementOfLiabilities" xlink:label="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockForSettlementOfLiabilities_2262" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockForSettlementOfLiabilitiesInShares" xlink:label="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockForSettlementOfLiabilitiesInShares_2268" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockAndCommonStockInConnectionWithG3CallOptionIssuance" xlink:label="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockAndCommonStockInConnectionWithG3CallOptionIssuance_2272" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_1079_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain_1085_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain_1085" xlink:to="us-gaap_PreferredStockMember_1081" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain_1085" xlink:to="us-gaap_CommonStockMember_1082" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain_1085" xlink:to="us-gaap_AdditionalPaidInCapitalMember_1083" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain_1085" xlink:to="us-gaap_RetainedEarningsMember_1084" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1079" xlink:to="us-gaap_SeriesAPreferredStockMember_1076" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1079" xlink:to="us-gaap_SeriesBPreferredStockMember_1077" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1079" xlink:to="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2457" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_1079" xlink:to="us-gaap_SeriesCPreferredStockMember_1078" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalWarrantIssued_2008" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_AdjustmentsToAdditionalPaidInCapitalExciseTaxPayableAttributableToRedemptionOfCommonStock_2009" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_1075" xlink:to="us-gaap_ClassOfStockDomain_1079" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable_1074" xlink:to="us-gaap_StatementEquityComponentsAxis_1080" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StockIssuedDuringPeriodSharesOther_1092" />
    <link:definitionArc xlink:type="arc" order="40.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_1093" />
    <link:definitionArc xlink:type="arc" order="41.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_NetIncomeLoss_1094" />
    <link:definitionArc xlink:type="arc" order="42.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StockholdersEquity_1095" />
    <link:definitionArc xlink:type="arc" order="43.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_SharesOutstanding_1096" />
    <link:definitionArc xlink:type="arc" order="23.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfSharesInConnectionWithTheConversionOfTheRelatedPartyConvertiblePromissoryNote_2284" />
    <link:definitionArc xlink:type="arc" order="24.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfSharesInConnectionWithTheConversionOfTheRelatedPartyConvertiblePromissoryNoteInShares_2285" />
    <link:definitionArc xlink:type="arc" order="25.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfSharesInConnectionWithTheConversionOfThePipeNote_2289" />
    <link:definitionArc xlink:type="arc" order="26.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfSharesInConnectionWithTheConversionOfThePipeNoteInShares_2291" />
    <link:definitionArc xlink:type="arc" order="27.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_CancellationOfFractionalSharesInConnectionWithAugustReverseStockSplit_2296" />
    <link:definitionArc xlink:type="arc" order="28.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_CancellationOfFractionalSharesInConnectionWithAugustReverseStockSplitInShares_2297" />
    <link:definitionArc xlink:type="arc" order="29.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities_2330" />
    <link:definitionArc xlink:type="arc" order="30.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_2331" />
    <link:definitionArc xlink:type="arc" order="31.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StockIssuedDuringPeriodValueEmployeeStockOwnershipPlan_2332" />
    <link:definitionArc xlink:type="arc" order="32.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockOwnershipPlan_2333" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StatementTable_1074" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable_1074" xlink:to="us-gaap_StatementClassOfStockAxis_1075" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis_1080" xlink:to="us-gaap_EquityComponentDomain_1085" />
    <link:definitionArc xlink:type="arc" order="33.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_ConversionOfDebtToCommonSharesInConnectionWithTheMerger_2336" />
    <link:definitionArc xlink:type="arc" order="34.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_ConversionOfDebtToCommonSharesInConnectionWithTheMergerInShares_2360" />
    <link:definitionArc xlink:type="arc" order="35.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfCommonStockToNorthviewStockholdersAsAResultOfTheMerger_2367" />
    <link:definitionArc xlink:type="arc" order="36.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfCommonStockToNorthvieStockholdersAsAResultOfTheMerger_2368" />
    <link:definitionArc xlink:type="arc" order="37.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfInducedCommonSharesToRelatedPartyInConnectionWithTheMerger_2369" />
    <link:definitionArc xlink:type="arc" order="39.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_StockIssuedDuringPeriodValueIssuanceOfWarrantsToFinancialAdvisorInConnectionWithTheMerger_2458" />
    <link:definitionArc xlink:type="arc" order="38.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_StockIssuedDuringPeriodSharesIssuanceOfInducedCommonSharesToRelatedPartyInConnectionWithTheMerger_2459" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues_1089" />
    <link:definitionArc xlink:type="arc" order="22.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockAndCommonStockInConnectionWithG3CallOptionIssuanceInShares_2277" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues_1090" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="us-gaap_StockIssuedDuringPeriodValueOther_1091" />
    <link:definitionArc xlink:type="arc" order="19.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockForSettlementOfLiabilities_2262" />
    <link:definitionArc xlink:type="arc" order="20.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockForSettlementOfLiabilitiesInShares_2268" />
    <link:definitionArc xlink:type="arc" order="21.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1086" xlink:to="pfsa_IssuanceOfSeriesANon-VotingConvertiblePreferredStockAndCommonStockInConnectionWithG3CallOptionIssuance_2272" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_1075" xlink:to="us-gaap_ClassOfStockDomain_1079_default" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis_1080" xlink:to="us-gaap_EquityComponentDomain_1085_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/CondensedConsolidatedBalanceSheets" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyMember" xlink:label="us-gaap_RelatedPartyMember_1006" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:label="us-gaap_PrepaidExpenseAndOtherAssetsCurrent_1010" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CryptoAssetFairValueCurrent" xlink:label="us-gaap_CryptoAssetFairValueCurrent_1011" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsCurrent" xlink:label="us-gaap_AssetsCurrent_1012" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="us-gaap_PropertyPlantAndEquipmentNet_1013" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherAssetsNoncurrent" xlink:label="us-gaap_OtherAssetsNoncurrent_1014" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DeferredCosts" xlink:label="us-gaap_DeferredCosts_1015" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Assets" xlink:label="us-gaap_Assets_1016" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherLoansPayableCurrent" xlink:label="us-gaap_OtherLoansPayableCurrent_1025" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesCurrentAbstract" xlink:label="us-gaap_LiabilitiesCurrentAbstract_1017" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsPayableCurrent" xlink:label="us-gaap_AccountsPayableCurrent_1018" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SalesAndExciseTaxPayableCurrent" xlink:label="us-gaap_SalesAndExciseTaxPayableCurrent_1019" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:label="us-gaap_AccruedLiabilitiesCurrent_1020" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherLiabilitiesCurrent" xlink:label="us-gaap_OtherLiabilitiesCurrent_1021" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleDebtCurrent" xlink:label="us-gaap_ConvertibleDebtCurrent_1022" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NotesPayableCurrent" xlink:label="us-gaap_NotesPayableCurrent_1023" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ConvertibleAndPromissoryNotesPayable" xlink:label="pfsa_ConvertibleAndPromissoryNotesPayable_1024" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockValue" xlink:label="us-gaap_CommonStockValue_1033" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementOfFinancialPositionAbstract" xlink:label="us-gaap_StatementOfFinancialPositionAbstract_1002" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable_1003" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1004" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_1005" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems_1007" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsCurrentAbstract" xlink:label="us-gaap_AssetsCurrentAbstract_1008" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapital" xlink:label="us-gaap_AdditionalPaidInCapital_1034" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit_1035" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity_1036" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesAndStockholdersEquity" xlink:label="us-gaap_LiabilitiesAndStockholdersEquity_1037" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsAbstract" xlink:label="us-gaap_AssetsAbstract_2235" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:label="us-gaap_LiabilitiesAndStockholdersEquityAbstract_2236" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_CallOptionAsset" xlink:label="pfsa_CallOptionAsset_2238" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesCurrent" xlink:label="us-gaap_LiabilitiesCurrent_1026" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents_2084" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstanding" xlink:label="us-gaap_WarrantsAndRightsOutstanding_1027" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_LoansPayableRelatedPartyAtFairValue" xlink:label="pfsa_LoansPayableRelatedPartyAtFairValue_1028" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Liabilities" xlink:label="us-gaap_Liabilities_1029" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommitmentsAndContingencies" xlink:label="us-gaap_CommitmentsAndContingencies_1030" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityAbstract" xlink:label="us-gaap_StockholdersEquityAbstract_1031" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockValue" xlink:label="us-gaap_PreferredStockValue_1032" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_1005_default" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain_1005" xlink:to="us-gaap_RelatedPartyMember_1006" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract_1008" xlink:to="us-gaap_PrepaidExpenseAndOtherAssetsCurrent_1010" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract_1008" xlink:to="us-gaap_CryptoAssetFairValueCurrent_1011" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract_1008" xlink:to="us-gaap_AssetsCurrent_1012" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract_2235" xlink:to="us-gaap_PropertyPlantAndEquipmentNet_1013" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract_2235" xlink:to="us-gaap_OtherAssetsNoncurrent_1014" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract_2235" xlink:to="us-gaap_DeferredCosts_1015" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract_2235" xlink:to="us-gaap_Assets_1016" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1017" xlink:to="us-gaap_OtherLoansPayableCurrent_1025" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract_2236" xlink:to="us-gaap_LiabilitiesCurrentAbstract_1017" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1017" xlink:to="us-gaap_AccountsPayableCurrent_1018" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1017" xlink:to="us-gaap_SalesAndExciseTaxPayableCurrent_1019" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1017" xlink:to="us-gaap_AccruedLiabilitiesCurrent_1020" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1017" xlink:to="us-gaap_OtherLiabilitiesCurrent_1021" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1017" xlink:to="us-gaap_ConvertibleDebtCurrent_1022" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1017" xlink:to="us-gaap_NotesPayableCurrent_1023" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1017" xlink:to="pfsa_ConvertibleAndPromissoryNotesPayable_1024" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract_1031" xlink:to="us-gaap_CommonStockValue_1033" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems_1007" xlink:to="us-gaap_StatementTable_1003" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable_1003" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1004" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1004" xlink:to="us-gaap_RelatedPartyDomain_1005" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract_2235" xlink:to="us-gaap_AssetsCurrentAbstract_1008" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract_1031" xlink:to="us-gaap_AdditionalPaidInCapital_1034" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract_1031" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit_1035" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract_1031" xlink:to="us-gaap_StockholdersEquity_1036" />
    <link:definitionArc xlink:type="arc" order="24.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1007" xlink:to="us-gaap_LiabilitiesAndStockholdersEquity_1037" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1007" xlink:to="us-gaap_AssetsAbstract_2235" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1007" xlink:to="us-gaap_LiabilitiesAndStockholdersEquityAbstract_2236" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract_2235" xlink:to="pfsa_CallOptionAsset_2238" />
    <link:definitionArc xlink:type="arc" order="18.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract_1017" xlink:to="us-gaap_LiabilitiesCurrent_1026" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract_1008" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents_2084" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract_2236" xlink:to="us-gaap_WarrantsAndRightsOutstanding_1027" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract_2236" xlink:to="pfsa_LoansPayableRelatedPartyAtFairValue_1028" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract_2236" xlink:to="us-gaap_Liabilities_1029" />
    <link:definitionArc xlink:type="arc" order="22.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1007" xlink:to="us-gaap_CommitmentsAndContingencies_1030" />
    <link:definitionArc xlink:type="arc" order="23.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_1007" xlink:to="us-gaap_StockholdersEquityAbstract_1031" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract_1031" xlink:to="us-gaap_PreferredStockValue_1032" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_1004" xlink:to="us-gaap_RelatedPartyDomain_1005_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/FairValueMeasurementScheduleofKeyInputsintotheMonteCarloSimulationModelfortheConvertiblePromissoryNoteDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2302" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember_2303" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember_2304" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConvertibleDebtMember" xlink:label="us-gaap_ConvertibleDebtMember_2447" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputExpectedTermMember" xlink:label="us-gaap_MeasurementInputExpectedTermMember_2118" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputOptionVolatilityMember" xlink:label="us-gaap_MeasurementInputOptionVolatilityMember_2119" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueOfCommonStockMember" xlink:label="pfsa_FairValueOfCommonStockMember_2120" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_MeasurementInputDebtRateMember" xlink:label="pfsa_MeasurementInputDebtRateMember_2117" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis_2301" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtTypeAxis" xlink:label="us-gaap_ShortTermDebtTypeAxis_2445" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtTypeDomain" xlink:label="us-gaap_ShortTermDebtTypeDomain_2446" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_FairValueMeasurementScheduleKeyInputsIntoTheMonteCarloSimulationModelForTheConvertiblePromissoryNoteAbstract" xlink:label="pfsa_FairValueMeasurementScheduleKeyInputsIntoTheMonteCarloSimulationModelForTheConvertiblePromissoryNoteAbstract_2095" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfKeyInputsIntoTheMonteCarloSimulationModelForTheConvertiblePromissoryNoteLineItems" xlink:label="pfsa_ScheduleOfKeyInputsIntoTheMonteCarloSimulationModelForTheConvertiblePromissoryNoteLineItems_2121" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentMeasurementInput" xlink:label="us-gaap_DebtInstrumentMeasurementInput_2122" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_2096" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="us-gaap_MeasurementInputTypeAxis_2100" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain_2101" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember_2302_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtTypeDomain" xlink:label="us-gaap_ShortTermDebtTypeDomain_2446_default" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain_2101_default" />
    <link:definitionArc xlink:type="arc" order="332.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis_2301" xlink:to="srt_RangeMember_2302" />
    <link:definitionArc xlink:type="arc" order="333.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2302" xlink:to="srt_MinimumMember_2303" />
    <link:definitionArc xlink:type="arc" order="334.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember_2302" xlink:to="srt_MaximumMember_2304" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShortTermDebtTypeDomain_2446" xlink:to="us-gaap_ConvertibleDebtMember_2447" />
    <link:definitionArc xlink:type="arc" order="202.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2101" xlink:to="us-gaap_MeasurementInputExpectedTermMember_2118" />
    <link:definitionArc xlink:type="arc" order="203.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2101" xlink:to="us-gaap_MeasurementInputOptionVolatilityMember_2119" />
    <link:definitionArc xlink:type="arc" order="204.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2101" xlink:to="pfsa_FairValueOfCommonStockMember_2120" />
    <link:definitionArc xlink:type="arc" order="201.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain_2101" xlink:to="pfsa_MeasurementInputDebtRateMember_2117" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_2096" xlink:to="srt_RangeAxis_2301" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_2096" xlink:to="us-gaap_ShortTermDebtTypeAxis_2445" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ShortTermDebtTypeAxis_2445" xlink:to="us-gaap_ShortTermDebtTypeDomain_2446" />
    <link:definitionArc xlink:type="arc" order="206.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="pfsa_ScheduleOfKeyInputsIntoTheMonteCarloSimulationModelForTheConvertiblePromissoryNoteLineItems_2121" xlink:to="us-gaap_DebtInstrumentMeasurementInput_2122" />
    <link:definitionArc xlink:type="arc" order="181.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="pfsa_ScheduleOfKeyInputsIntoTheMonteCarloSimulationModelForTheConvertiblePromissoryNoteLineItems_2121" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_2096" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_2096" xlink:to="us-gaap_MeasurementInputTypeAxis_2100" />
    <link:definitionArc xlink:type="arc" order="186.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_MeasurementInputTypeAxis_2100" xlink:to="us-gaap_MeasurementInputTypeDomain_2101" />
    <link:definitionArc xlink:type="arc" order="332.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis_2301" xlink:to="srt_RangeMember_2302_default" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ShortTermDebtTypeAxis_2445" xlink:to="us-gaap_ShortTermDebtTypeDomain_2446_default" />
    <link:definitionArc xlink:type="arc" order="186.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_MeasurementInputTypeAxis_2100" xlink:to="us-gaap_MeasurementInputTypeDomain_2101_default" />
  </link:definitionLink>
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/CondensedConsolidatedBalanceSheetsParentheticals" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_SeriesANon-VotingConvertiblePreferredStockMember" xlink:label="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2417" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable_2412" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis_2413" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_2414" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems_2415" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="us-gaap_PreferredStockSharesAuthorized_1041" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesIssued" xlink:label="us-gaap_PreferredStockSharesIssued_1042" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="us-gaap_PreferredStockSharesOutstanding_1043" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare_1044" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized_1045" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued_1046" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="us-gaap_CommonStockSharesOutstanding_1047" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementOfFinancialPositionAbstract" xlink:label="us-gaap_StatementOfFinancialPositionAbstract_1039" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="us-gaap_PreferredStockParOrStatedValuePerShare_1040" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_2414_default" />
    <link:definitionArc xlink:type="arc" order="424.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain_2414" xlink:to="pfsa_SeriesANon-VotingConvertiblePreferredStockMember_2417" />
    <link:definitionArc xlink:type="arc" order="419.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems_2415" xlink:to="us-gaap_StatementTable_2412" />
    <link:definitionArc xlink:type="arc" order="420.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable_2412" xlink:to="us-gaap_StatementClassOfStockAxis_2413" />
    <link:definitionArc xlink:type="arc" order="421.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis_2413" xlink:to="us-gaap_ClassOfStockDomain_2414" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_2415" xlink:to="us-gaap_PreferredStockSharesAuthorized_1041" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_2415" xlink:to="us-gaap_PreferredStockSharesIssued_1042" />
    <link:definitionArc xlink:type="arc" order="13.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_2415" xlink:to="us-gaap_PreferredStockSharesOutstanding_1043" />
    <link:definitionArc xlink:type="arc" order="14.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_2415" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare_1044" />
    <link:definitionArc xlink:type="arc" order="15.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_2415" xlink:to="us-gaap_CommonStockSharesAuthorized_1045" />
    <link:definitionArc xlink:type="arc" order="16.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_2415" xlink:to="us-gaap_CommonStockSharesIssued_1046" />
    <link:definitionArc xlink:type="arc" order="17.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_2415" xlink:to="us-gaap_CommonStockSharesOutstanding_1047" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems_2415" xlink:to="us-gaap_PreferredStockParOrStatedValuePerShare_1040" />
    <link:definitionArc xlink:type="arc" order="421.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis_2413" xlink:to="us-gaap_ClassOfStockDomain_2414_default" />
  </link:definitionLink>
  <link:roleRef roleURI="http://profusa.com/20260930/role/StockOptionPlanScheduleofStockCompensationExpenseDetails1" xlink:href="pfsa-20260930.xsd#StockOptionPlanScheduleofStockCompensationExpenseDetails1" xlink:type="simple" />
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/StockOptionPlanScheduleofStockCompensationExpenseDetails1" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementOfIncomeLocationBalanceAxis" xlink:label="us-gaap_StatementOfIncomeLocationBalanceAxis_2477" />
    <link:loc xlink:type="locator" xlink:href="pfsa-20260930.xsd#pfsa_ScheduleOfStockCompensationExpenseAbstract" xlink:label="pfsa_ScheduleOfStockCompensationExpenseAbstract_1383" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_1384" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_1389" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense_1390" />
    <link:definitionArc xlink:type="arc" order="12.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_1384" xlink:to="us-gaap_StatementOfIncomeLocationBalanceAxis_2477" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_1389" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_1384" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_1389" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense_1390" />
  </link:definitionLink>
  <link:roleRef roleURI="http://profusa.com/20260930/role/StockOptionPlanScheduleofStockCompensationExpenseDetails" xlink:href="pfsa-20260930.xsd#StockOptionPlanScheduleofStockCompensationExpenseDetails" xlink:type="simple" />
  <link:definitionLink xlink:role="http://profusa.com/20260930/role/StockOptionPlanScheduleofStockCompensationExpenseDetails" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_1384" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_1389" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense_1390" />
    <link:definitionArc xlink:type="arc" order="11.00000" priority="1" xbrldt:closed="true" use="optional" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_1389" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_1384" />
    <link:definitionArc xlink:type="arc" order="10.00000" priority="1" xbrldt:closed="false" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_1389" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense_1390" />
  </link:definitionLink>
  <link:roleRef roleURI="http://fasb.org/srt/role/srt-eedm/ExtensibleEnumerationLists" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-roles-2026.xsd#eedm1" xlink:type="simple" />
  <link:definitionLink xlink:role="http://fasb.org/srt/role/srt-eedm/ExtensibleEnumerationLists" xlink:type="extended" xlink:title="definitionLink">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ChiefExecutiveOfficerMember" xlink:label="srt_ChiefExecutiveOfficerMember" />
    <link:definitionArc xlink:type="arc" order="0" priority="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:to="srt_ChiefExecutiveOfficerMember" />
  </link:definitionLink>
</link:linkbase>
