<?xml version="1.0" encoding="UTF-8"?>

<!--XBRL Document Created with the Workiva Platform-->
<!--Copyright 2026 Workiva-->
<!--r:019d02fd-4baf-7903-b4f2-04ffc66c3b0f,g:46be0b7b-7401-4059-b553-d3752e346105-->
<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:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member"/>
  <link:roleRef roleURI="http://docusign.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFOPERATIONSANDCOMPREHENSIVEINCOMEUnaudited" xlink:type="simple" xlink:href="docu-20260430.xsd#CONDENSEDCONSOLIDATEDSTATEMENTSOFOPERATIONSANDCOMPREHENSIVEINCOMEUnaudited"/>
  <link:definitionLink xlink:role="http://docusign.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFOPERATIONSANDCOMPREHENSIVEINCOMEUnaudited" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_019d02fd-4c85-7c99-9286-161f7ccbed39" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_StatementTable_019d02fd-4c85-7c99-9286-161f7ccbed39" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationAxis_019d02fd-4c85-74df-b602-b953eb23fb52" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_019d02fd-4c85-7c99-9286-161f7ccbed39" xlink:to="loc_us-gaap_IncomeStatementLocationAxis_019d02fd-4c85-74df-b602-b953eb23fb52" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationDomain_019d02fd-4c85-74df-b602-b953eb23fb52_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_IncomeStatementLocationAxis_019d02fd-4c85-74df-b602-b953eb23fb52" xlink:to="loc_us-gaap_IncomeStatementLocationDomain_019d02fd-4c85-74df-b602-b953eb23fb52_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationDomain_019d02fd-4c85-79a0-aec7-d146c38ba990" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_IncomeStatementLocationAxis_019d02fd-4c85-74df-b602-b953eb23fb52" xlink:to="loc_us-gaap_IncomeStatementLocationDomain_019d02fd-4c85-79a0-aec7-d146c38ba990" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CostOfSalesMember_019d02fd-4c85-70a4-bcd8-483a913800b9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CostOfSalesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_019d02fd-4c85-79a0-aec7-d146c38ba990" xlink:to="loc_us-gaap_CostOfSalesMember_019d02fd-4c85-70a4-bcd8-483a913800b9" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SellingAndMarketingExpenseMember_019d02fd-4c85-73c7-b25f-793c93d88b8e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SellingAndMarketingExpenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_019d02fd-4c85-79a0-aec7-d146c38ba990" xlink:to="loc_us-gaap_SellingAndMarketingExpenseMember_019d02fd-4c85-73c7-b25f-793c93d88b8e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ResearchAndDevelopmentExpenseMember_019d02fd-4c85-7f52-9b08-5105f63e3175" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_019d02fd-4c85-79a0-aec7-d146c38ba990" xlink:to="loc_us-gaap_ResearchAndDevelopmentExpenseMember_019d02fd-4c85-7f52-9b08-5105f63e3175" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GeneralAndAdministrativeExpenseMember_019d02fd-4c85-74f2-8e5c-51e252467f76" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_019d02fd-4c85-79a0-aec7-d146c38ba990" xlink:to="loc_us-gaap_GeneralAndAdministrativeExpenseMember_019d02fd-4c85-74f2-8e5c-51e252467f76" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019d02fd-4c85-740b-8866-dbbdbc1ca742" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019d02fd-4c85-740b-8866-dbbdbc1ca742" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CostOfGoodsAndServicesSold_019d02fd-4c85-7c32-aa27-5beaffad2695" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CostOfGoodsAndServicesSold"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_CostOfGoodsAndServicesSold_019d02fd-4c85-7c32-aa27-5beaffad2695" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GrossProfit_019d02fd-4c85-7191-8379-50c4a0b9ced9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GrossProfit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_GrossProfit_019d02fd-4c85-7191-8379-50c4a0b9ced9" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingExpensesAbstract_019d02fd-4c85-7a5c-b6d8-098b3a3fa21d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingExpensesAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_OperatingExpensesAbstract_019d02fd-4c85-7a5c-b6d8-098b3a3fa21d" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SellingAndMarketingExpense_019d02fd-4c85-7f6e-9a9e-c62292e09caf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SellingAndMarketingExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_019d02fd-4c85-7a5c-b6d8-098b3a3fa21d" xlink:to="loc_us-gaap_SellingAndMarketingExpense_019d02fd-4c85-7f6e-9a9e-c62292e09caf" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ResearchAndDevelopmentExpense_019d02fd-4c85-73c7-a73a-713f48fae25b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_019d02fd-4c85-7a5c-b6d8-098b3a3fa21d" xlink:to="loc_us-gaap_ResearchAndDevelopmentExpense_019d02fd-4c85-73c7-a73a-713f48fae25b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GeneralAndAdministrativeExpense_019d02fd-4c85-7fd4-8af3-aef501b38026" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_019d02fd-4c85-7a5c-b6d8-098b3a3fa21d" xlink:to="loc_us-gaap_GeneralAndAdministrativeExpense_019d02fd-4c85-7fd4-8af3-aef501b38026" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingExpenses_019d02fd-4c85-7f71-80bf-93e2255024cf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingExpenses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_019d02fd-4c85-7a5c-b6d8-098b3a3fa21d" xlink:to="loc_us-gaap_OperatingExpenses_019d02fd-4c85-7f71-80bf-93e2255024cf" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingIncomeLoss_019d02fd-4c85-7cdc-8d06-4f65571c0804" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_OperatingIncomeLoss_019d02fd-4c85-7cdc-8d06-4f65571c0804" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestExpenseNonoperating_019d02fd-4c85-7f33-a3bd-697ca3421149" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InterestExpenseNonoperating"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_InterestExpenseNonoperating_019d02fd-4c85-7f33-a3bd-697ca3421149" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NonoperatingIncomeExpense_019d02fd-4c85-72f0-ad83-77d3b22d3fce" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NonoperatingIncomeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_NonoperatingIncomeExpense_019d02fd-4c85-72f0-ad83-77d3b22d3fce" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest_019d02fd-4c85-7735-a95d-d763a33d9727" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest_019d02fd-4c85-7735-a95d-d763a33d9727" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeTaxExpenseBenefit_019d02fd-4c85-7e4f-9c88-b6573475f73b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxExpenseBenefit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_IncomeTaxExpenseBenefit_019d02fd-4c85-7e4f-9c88-b6573475f73b" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_019d02fd-4c85-77da-bebc-609a6bd99d43" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_NetIncomeLoss_019d02fd-4c85-77da-bebc-609a6bd99d43" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EarningsPerShareAbstract_019d02fd-4c85-7bc8-b824-98a371cfa10d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EarningsPerShareAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_EarningsPerShareAbstract_019d02fd-4c85-7bc8-b824-98a371cfa10d" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EarningsPerShareBasic_019d02fd-4c85-7b18-9217-7fe544fd0076" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EarningsPerShareBasic"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EarningsPerShareAbstract_019d02fd-4c85-7bc8-b824-98a371cfa10d" xlink:to="loc_us-gaap_EarningsPerShareBasic_019d02fd-4c85-7b18-9217-7fe544fd0076" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EarningsPerShareDiluted_019d02fd-4c85-7468-88cd-ed2f7efb5813" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EarningsPerShareDiluted"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EarningsPerShareAbstract_019d02fd-4c85-7bc8-b824-98a371cfa10d" xlink:to="loc_us-gaap_EarningsPerShareDiluted_019d02fd-4c85-7468-88cd-ed2f7efb5813" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WeightedAverageNumberOfSharesOutstandingDilutedDisclosureItemsAbstract_019d02fd-4c85-7c0b-9503-68e75f411432" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingDilutedDisclosureItemsAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_WeightedAverageNumberOfSharesOutstandingDilutedDisclosureItemsAbstract_019d02fd-4c85-7c0b-9503-68e75f411432" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WeightedAverageNumberOfSharesOutstandingBasic_019d02fd-4c85-7f17-9e89-d1919bddd4c1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingBasic"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_WeightedAverageNumberOfSharesOutstandingDilutedDisclosureItemsAbstract_019d02fd-4c85-7c0b-9503-68e75f411432" xlink:to="loc_us-gaap_WeightedAverageNumberOfSharesOutstandingBasic_019d02fd-4c85-7f17-9e89-d1919bddd4c1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WeightedAverageNumberOfDilutedSharesOutstanding_019d02fd-4c85-7655-82e7-16e9bf23ecac" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WeightedAverageNumberOfDilutedSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_WeightedAverageNumberOfSharesOutstandingDilutedDisclosureItemsAbstract_019d02fd-4c85-7c0b-9503-68e75f411432" xlink:to="loc_us-gaap_WeightedAverageNumberOfDilutedSharesOutstanding_019d02fd-4c85-7655-82e7-16e9bf23ecac" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParentAbstract_019d02fd-4c85-7443-bfa0-ddd500ece90e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParentAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParentAbstract_019d02fd-4c85-7443-bfa0-ddd500ece90e" xlink:type="arc" order="12"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent_019d02fd-4c85-7f60-92e5-c832f193f59c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParentAbstract_019d02fd-4c85-7443-bfa0-ddd500ece90e" xlink:to="loc_us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent_019d02fd-4c85-7f60-92e5-c832f193f59c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeUnrealizedHoldingGainLossOnSecuritiesArisingDuringPeriodNetOfTax_019d02fd-4c85-7270-b726-9932626523c3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeUnrealizedHoldingGainLossOnSecuritiesArisingDuringPeriodNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParentAbstract_019d02fd-4c85-7443-bfa0-ddd500ece90e" xlink:to="loc_us-gaap_OtherComprehensiveIncomeUnrealizedHoldingGainLossOnSecuritiesArisingDuringPeriodNetOfTax_019d02fd-4c85-7270-b726-9932626523c3" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_019d02fd-4c85-71b3-8e74-e75cf7de5d45" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParentAbstract_019d02fd-4c85-7443-bfa0-ddd500ece90e" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_019d02fd-4c85-71b3-8e74-e75cf7de5d45" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComprehensiveIncomeNetOfTax_019d02fd-4c85-7f4d-94df-ce8fc2702ecd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ComprehensiveIncomeNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_us-gaap_ComprehensiveIncomeNetOfTax_019d02fd-4c85-7f4d-94df-ce8fc2702ecd" xlink:type="arc" order="13"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_AllocatedSharebasedCompensationExpenseAbstract_019d02fd-4c85-7e02-a162-9078cf57266a" xlink:href="docu-20260430.xsd#docu_AllocatedSharebasedCompensationExpenseAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-7f8e-b0dd-c8753fdc0703" xlink:to="loc_docu_AllocatedSharebasedCompensationExpenseAbstract_019d02fd-4c85-7e02-a162-9078cf57266a" xlink:type="arc" order="14"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_019d02fd-4c85-7a2d-930b-90e6495244e0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_docu_AllocatedSharebasedCompensationExpenseAbstract_019d02fd-4c85-7e02-a162-9078cf57266a" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_019d02fd-4c85-7a2d-930b-90e6495244e0" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFSTOCKHOLDERSEQUITYUnaudited" xlink:type="simple" xlink:href="docu-20260430.xsd#CONDENSEDCONSOLIDATEDSTATEMENTSOFSTOCKHOLDERSEQUITYUnaudited"/>
  <link:definitionLink xlink:role="http://docusign.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFSTOCKHOLDERSEQUITYUnaudited" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_019d02fd-4c85-718f-91b0-23414d253291" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_019d02fd-4c85-764e-a9f7-e9be7f84fe12" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-718f-91b0-23414d253291" xlink:to="loc_us-gaap_StatementTable_019d02fd-4c85-764e-a9f7-e9be7f84fe12" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_019d02fd-4c85-77b5-b2d7-265e5b766195" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_019d02fd-4c85-764e-a9f7-e9be7f84fe12" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_019d02fd-4c85-77b5-b2d7-265e5b766195" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019d02fd-4c85-77b5-b2d7-265e5b766195_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019d02fd-4c85-77b5-b2d7-265e5b766195" xlink:to="loc_us-gaap_EquityComponentDomain_019d02fd-4c85-77b5-b2d7-265e5b766195_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019d02fd-4c85-7afe-bb4e-db54b7ebea75" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019d02fd-4c85-77b5-b2d7-265e5b766195" xlink:to="loc_us-gaap_EquityComponentDomain_019d02fd-4c85-7afe-bb4e-db54b7ebea75" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockMember_019d02fd-4c85-707b-9c65-a46ff2aba95f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019d02fd-4c85-7afe-bb4e-db54b7ebea75" xlink:to="loc_us-gaap_CommonStockMember_019d02fd-4c85-707b-9c65-a46ff2aba95f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdditionalPaidInCapitalMember_019d02fd-4c85-79b1-a2e8-0974d48bbb9d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdditionalPaidInCapitalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019d02fd-4c85-7afe-bb4e-db54b7ebea75" xlink:to="loc_us-gaap_AdditionalPaidInCapitalMember_019d02fd-4c85-79b1-a2e8-0974d48bbb9d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TreasuryStockCommonMember_019d02fd-4c85-7cf8-a3bd-233a108c2aab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TreasuryStockCommonMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019d02fd-4c85-7afe-bb4e-db54b7ebea75" xlink:to="loc_us-gaap_TreasuryStockCommonMember_019d02fd-4c85-7cf8-a3bd-233a108c2aab" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019d02fd-4c85-7e6a-9f25-7446695f26b1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019d02fd-4c85-7afe-bb4e-db54b7ebea75" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019d02fd-4c85-7e6a-9f25-7446695f26b1" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsMember_019d02fd-4c85-758b-82ae-a755d1c8dc71" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019d02fd-4c85-7afe-bb4e-db54b7ebea75" xlink:to="loc_us-gaap_RetainedEarningsMember_019d02fd-4c85-758b-82ae-a755d1c8dc71" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019d02fd-4c85-718f-91b0-23414d253291" xlink:to="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_019d02fd-4c85-7496-9141-9c3044d11109" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_CommonStockSharesOutstanding_019d02fd-4c85-7496-9141-9c3044d11109" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_019d02fd-4c85-77a1-a2c3-3e06a086b676" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_StockholdersEquity_019d02fd-4c85-77a1-a2c3-3e06a086b676" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_019d02fd-4c85-7ddf-b36a-70474f1500ab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_019d02fd-4c85-7ddf-b36a-70474f1500ab" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised_019d02fd-4c85-7a91-bb0f-adcf892c3e11" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised_019d02fd-4c85-7a91-bb0f-adcf892c3e11" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures_019d02fd-4c85-7cf1-a71c-d70be53ecb2a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures_019d02fd-4c85-7cf1-a71c-d70be53ecb2a" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_019d02fd-4c85-7961-acd0-10e87598427c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_019d02fd-4c85-7961-acd0-10e87598427c" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan_019d02fd-4c85-79fb-aefe-68bc82b6769e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan_019d02fd-4c85-79fb-aefe-68bc82b6769e" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharesPaidForTaxWithholdingForShareBasedCompensation_019d02fd-4c85-778a-b0a8-9275022505a0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharesPaidForTaxWithholdingForShareBasedCompensation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_SharesPaidForTaxWithholdingForShareBasedCompensation_019d02fd-4c85-778a-b0a8-9275022505a0" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsRelatedToTaxWithholdingForShareBasedCompensation_019d02fd-4c85-7e1f-85bb-e73a80218a59" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsRelatedToTaxWithholdingForShareBasedCompensation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_AdjustmentsRelatedToTaxWithholdingForShareBasedCompensation_019d02fd-4c85-7e1f-85bb-e73a80218a59" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TreasuryStockRetiredCostMethodAmount_019e2a2b-692a-715a-8535-8a64afe299b4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TreasuryStockRetiredCostMethodAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_TreasuryStockRetiredCostMethodAmount_019e2a2b-692a-715a-8535-8a64afe299b4" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodShares_019d02fd-4c85-7dc6-9a5b-7dfc669f6390" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockRepurchasedAndRetiredDuringPeriodShares"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodShares_019d02fd-4c85-7dc6-9a5b-7dfc669f6390" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodValue_019d02fd-4c85-7305-b546-b7348abbd106" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockRepurchasedAndRetiredDuringPeriodValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodValue_019d02fd-4c85-7305-b546-b7348abbd106" xlink:type="arc" order="12"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalShareBasedCompensationEmployeeStockPurchaseProgramRequisiteServicePeriodRecognition_019d02fd-4c85-7b05-ab49-772332f8c847" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalShareBasedCompensationEmployeeStockPurchaseProgramRequisiteServicePeriodRecognition"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalShareBasedCompensationEmployeeStockPurchaseProgramRequisiteServicePeriodRecognition_019d02fd-4c85-7b05-ab49-772332f8c847" xlink:type="arc" order="13"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_019d02fd-4c85-7d37-a094-938d892fd51d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_NetIncomeLoss_019d02fd-4c85-7d37-a094-938d892fd51d" xlink:type="arc" order="14"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_019d02fd-4c85-720a-9abb-fb79d3a62c71" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019d02fd-4c85-7fbe-b887-c2374bd18c97" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_019d02fd-4c85-720a-9abb-fb79d3a62c71" xlink:type="arc" order="15"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_019d02fd-4c85-7636-a3e7-aa1ce5d62976" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_019d02fd-4c85-7cd1-b76b-7823770b7dc6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/RevenueScheduleofRevenuebyRevenueTypeDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#RevenueScheduleofRevenuebyRevenueTypeDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/RevenueScheduleofRevenuebyRevenueTypeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueLineItems_019e1cc5-ed9f-7a44-bbd7-6d33684eb91d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueTable_019e1cc5-ed9f-7470-8afc-4cde2506befe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_019e1cc5-ed9f-7a44-bbd7-6d33684eb91d" xlink:to="loc_us-gaap_DisaggregationOfRevenueTable_019e1cc5-ed9f-7470-8afc-4cde2506befe" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_019e1cc5-ed9f-7060-ae9b-d5e91ca82d11" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_019e1cc5-ed9f-7470-8afc-4cde2506befe" xlink:to="loc_srt_ProductOrServiceAxis_019e1cc5-ed9f-7060-ae9b-d5e91ca82d11" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_019e1cc5-ed9f-7060-ae9b-d5e91ca82d11_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_019e1cc5-ed9f-7060-ae9b-d5e91ca82d11" xlink:to="loc_srt_ProductsAndServicesDomain_019e1cc5-ed9f-7060-ae9b-d5e91ca82d11_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_019e1cc5-ed9f-7a44-835b-8b8379d0d383" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_019e1cc5-ed9f-7060-ae9b-d5e91ca82d11" xlink:to="loc_srt_ProductsAndServicesDomain_019e1cc5-ed9f-7a44-835b-8b8379d0d383" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubscriptionAndCirculationMember_019e1cc5-ed9f-720e-a018-d5249ec2632d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubscriptionAndCirculationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_019e1cc5-ed9f-7a44-835b-8b8379d0d383" xlink:to="loc_us-gaap_SubscriptionAndCirculationMember_019e1cc5-ed9f-720e-a018-d5249ec2632d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_ProfessionalServicesAndOtherMember_019e1cc5-ed9f-735c-84bb-729ba7d152ff" xlink:href="docu-20260430.xsd#docu_ProfessionalServicesAndOtherMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_019e1cc5-ed9f-7a44-835b-8b8379d0d383" xlink:to="loc_docu_ProfessionalServicesAndOtherMember_019e1cc5-ed9f-735c-84bb-729ba7d152ff" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019e1cc5-ed9f-7aa7-93fa-fe8cfd842736" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_019e1cc5-ed9f-7a44-bbd7-6d33684eb91d" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019e1cc5-ed9f-7aa7-93fa-fe8cfd842736" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/RevenueNarrativeDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#RevenueNarrativeDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/RevenueNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019d02fd-4d50-7a56-b533-a38f7cee7d87" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019d02fd-4d50-7a56-b533-a38f7cee7d87" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionStartDateAxis_019d02fd-4d50-71ac-857c-b87a78f3b7c9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionStartDateAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019d02fd-4d50-7a56-b533-a38f7cee7d87" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionStartDateAxis_019d02fd-4d50-71ac-857c-b87a78f3b7c9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligation_019e1cca-c936-768e-91d8-c32852f609ab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligation_019e1cca-c936-768e-91d8-c32852f609ab" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationPercentage_019e1cca-c936-7404-a42d-bbf6b9cb20cb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligationPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationPercentage_019e1cca-c936-7404-a42d-bbf6b9cb20cb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1_019e1cca-c936-71b2-aff0-154f05481a92" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1_019e1cca-c936-71b2-aff0-154f05481a92" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerAssetNet_019e1cca-c936-7c16-971e-be968451f2ab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerAssetNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_ContractWithCustomerAssetNet_019e1cca-c936-7c16-971e-be968451f2ab" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerLiabilityRevenueRecognized_019e1cca-c936-710a-a102-bad6193bf9ab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerLiabilityRevenueRecognized"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_ContractWithCustomerLiabilityRevenueRecognized_019e1cca-c936-710a-a102-bad6193bf9ab" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/RevenueNarrativeDetails_1" xlink:type="simple" xlink:href="docu-20260430.xsd#RevenueNarrativeDetails_1"/>
  <link:definitionLink xlink:role="http://docusign.com/role/RevenueNarrativeDetails_1" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019d02fd-4d50-7a56-b533-a38f7cee7d87" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019d02fd-4d50-7a56-b533-a38f7cee7d87" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligation_019e1cca-c936-768e-91d8-c32852f609ab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligation_019e1cca-c936-768e-91d8-c32852f609ab" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationPercentage_019e1cca-c936-7404-a42d-bbf6b9cb20cb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligationPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationPercentage_019e1cca-c936-7404-a42d-bbf6b9cb20cb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1_019e1cca-c936-71b2-aff0-154f05481a92" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1_019e1cca-c936-71b2-aff0-154f05481a92" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerAssetNet_019e1cca-c936-7c16-971e-be968451f2ab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerAssetNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_ContractWithCustomerAssetNet_019e1cca-c936-7c16-971e-be968451f2ab" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerLiabilityRevenueRecognized_019e1cca-c936-710a-a102-bad6193bf9ab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerLiabilityRevenueRecognized"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019d02fd-4d50-73d9-b192-01bb8f17e3b9" xlink:to="loc_us-gaap_ContractWithCustomerLiabilityRevenueRecognized_019e1cca-c936-710a-a102-bad6193bf9ab" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/FairValueMeasurementsScheduleofAssetsandLiabilitiesMeasuredatFairValueonaRecurringBasisDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#FairValueMeasurementsScheduleofAssetsandLiabilitiesMeasuredatFairValueonaRecurringBasisDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/FairValueMeasurementsScheduleofAssetsandLiabilitiesMeasuredatFairValueonaRecurringBasisDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019d02fd-4d50-7b34-8b44-18c0b2a22af1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByBalanceSheetGroupingTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019d02fd-4d50-7b34-8b44-18c0b2a22af1" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019d02fd-4d50-7369-a90c-81f1565f1ff2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019d02fd-4d50-7b34-8b44-18c0b2a22af1" xlink:to="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019d02fd-4d50-7369-a90c-81f1565f1ff2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019d02fd-4d50-7369-a90c-81f1565f1ff2_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019d02fd-4d50-7369-a90c-81f1565f1ff2" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019d02fd-4d50-7369-a90c-81f1565f1ff2_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019d02fd-4d50-75b1-b6ec-0d7ceca08ac3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019d02fd-4d50-7369-a90c-81f1565f1ff2" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019d02fd-4d50-75b1-b6ec-0d7ceca08ac3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel1Member_019d02fd-4d50-7c1d-a6f9-18b2f026f705" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel1Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019d02fd-4d50-75b1-b6ec-0d7ceca08ac3" xlink:to="loc_us-gaap_FairValueInputsLevel1Member_019d02fd-4d50-7c1d-a6f9-18b2f026f705" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel2Member_019d02fd-4d50-7b26-853a-5b1bceb2d938" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019d02fd-4d50-75b1-b6ec-0d7ceca08ac3" xlink:to="loc_us-gaap_FairValueInputsLevel2Member_019d02fd-4d50-7b26-853a-5b1bceb2d938" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialInstrumentAxis_019d02fd-4d50-7965-be9a-30464b802d36" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019d02fd-4d50-7b34-8b44-18c0b2a22af1" xlink:to="loc_us-gaap_FinancialInstrumentAxis_019d02fd-4d50-7965-be9a-30464b802d36" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019d02fd-4d50-7965-be9a-30464b802d36_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FinancialInstrumentAxis_019d02fd-4d50-7965-be9a-30464b802d36" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019d02fd-4d50-7965-be9a-30464b802d36_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019d02fd-4d50-7052-b05f-0eef373c1612" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FinancialInstrumentAxis_019d02fd-4d50-7965-be9a-30464b802d36" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019d02fd-4d50-7052-b05f-0eef373c1612" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_019d02fd-4d50-7323-8f94-2dea55b0e208" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019d02fd-4d50-7052-b05f-0eef373c1612" xlink:to="loc_us-gaap_CommercialPaperMember_019d02fd-4d50-7323-8f94-2dea55b0e208" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CorporateNoteSecuritiesMember_019d02fd-4d50-7c70-bc0b-450adae7f25f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CorporateNoteSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019d02fd-4d50-7052-b05f-0eef373c1612" xlink:to="loc_us-gaap_CorporateNoteSecuritiesMember_019d02fd-4d50-7c70-bc0b-450adae7f25f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USGovernmentAgenciesDebtSecuritiesMember_019d02fd-4d50-7b82-a406-f0e5f409d6d1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USGovernmentAgenciesDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019d02fd-4d50-7052-b05f-0eef373c1612" xlink:to="loc_us-gaap_USGovernmentAgenciesDebtSecuritiesMember_019d02fd-4d50-7b82-a406-f0e5f409d6d1" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashEquivalentsAtCarryingValue_019e1cd0-5794-7d86-a11e-e288d8d9b5ba" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashEquivalentsAtCarryingValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_us-gaap_CashEquivalentsAtCarryingValue_019e1cd0-5794-7d86-a11e-e288d8d9b5ba" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_CashEquivalentsFairValueDisclosure_019e1cd0-c3c7-7c5d-853c-810b2de2cdbd" xlink:href="docu-20260430.xsd#docu_CashEquivalentsFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_docu_CashEquivalentsFairValueDisclosure_019e1cd0-c3c7-7c5d-853c-810b2de2cdbd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleAmortizedCostExcludingAccruedInterestAfterAllowanceForCreditLoss_019d02fd-4d50-7ddd-bb3d-02a771d284af" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleAmortizedCostExcludingAccruedInterestAfterAllowanceForCreditLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleAmortizedCostExcludingAccruedInterestAfterAllowanceForCreditLoss_019d02fd-4d50-7ddd-bb3d-02a771d284af" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax_019d02fd-4d50-79a5-ae58-5e50c1c4253a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax_019d02fd-4d50-79a5-ae58-5e50c1c4253a" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax_019d02fd-4d50-758a-bab6-a9000562745f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax_019d02fd-4d50-758a-bab6-a9000562745f" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleExcludingAccruedInterest_019d02fd-4d50-79be-842b-1e4afa5576aa" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleExcludingAccruedInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleExcludingAccruedInterest_019d02fd-4d50-79be-842b-1e4afa5576aa" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_CashEquivalentsAndDebtSecuritiesAvailableForSaleFairValue_019e1cd3-133c-7f45-9ab3-22aabadd6d72" xlink:href="docu-20260430.xsd#docu_CashEquivalentsAndDebtSecuritiesAvailableForSaleFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_docu_CashEquivalentsAndDebtSecuritiesAvailableForSaleFairValue_019e1cd3-133c-7f45-9ab3-22aabadd6d72" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_CashEquivalentsAndDebtSecuritiesAvailableForSaleAmortizedCost_019e1cd2-a78e-72fe-bb18-db59cfce567a" xlink:href="docu-20260430.xsd#docu_CashEquivalentsAndDebtSecuritiesAvailableForSaleAmortizedCost"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_docu_CashEquivalentsAndDebtSecuritiesAvailableForSaleAmortizedCost_019e1cd2-a78e-72fe-bb18-db59cfce567a" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_Cash_019d02fd-4d50-75b7-a847-8f72e9cce269" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_Cash"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-73e1-93ae-a9f9466b9e30" xlink:to="loc_us-gaap_Cash_019d02fd-4d50-75b7-a847-8f72e9cce269" xlink:type="arc" order="8"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/FairValueMeasurementsScheduleofFairValueofAvailableforsaleMarketableSecuritiesbyRemainingContractualMaturitiesDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#FairValueMeasurementsScheduleofFairValueofAvailableforsaleMarketableSecuritiesbyRemainingContractualMaturitiesDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/FairValueMeasurementsScheduleofFairValueofAvailableforsaleMarketableSecuritiesbyRemainingContractualMaturitiesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-7702-8c9b-183b56726d8b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019d02fd-4d50-701b-9713-b8e72b16a4a5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByBalanceSheetGroupingTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-7702-8c9b-183b56726d8b" xlink:to="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019d02fd-4d50-701b-9713-b8e72b16a4a5" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationAxis_019d02fd-4d50-7289-b98e-381294abb287" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019d02fd-4d50-701b-9713-b8e72b16a4a5" xlink:to="loc_us-gaap_BalanceSheetLocationAxis_019d02fd-4d50-7289-b98e-381294abb287" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_019d02fd-4d50-7289-b98e-381294abb287_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_019d02fd-4d50-7289-b98e-381294abb287" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_019d02fd-4d50-7289-b98e-381294abb287_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_019d02fd-4d50-701d-9ea1-733872f06777" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_019d02fd-4d50-7289-b98e-381294abb287" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_019d02fd-4d50-701d-9ea1-733872f06777" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShortTermInvestmentsMember_019d02fd-4d50-7612-9535-bac45e36b967" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShortTermInvestmentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BalanceSheetLocationDomain_019d02fd-4d50-701d-9ea1-733872f06777" xlink:to="loc_us-gaap_ShortTermInvestmentsMember_019d02fd-4d50-7612-9535-bac45e36b967" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesWithinOneYearFairValue_019d02fd-4d50-75e3-86f1-4595618b2dc4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesDebtMaturitiesWithinOneYearFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-7702-8c9b-183b56726d8b" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesWithinOneYearFairValue_019d02fd-4d50-75e3-86f1-4595618b2dc4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesAfterOneThroughFiveYearsFairValue_019d02fd-4d50-7101-a5fd-377a656cc1b4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesDebtMaturitiesAfterOneThroughFiveYearsFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-7702-8c9b-183b56726d8b" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesAfterOneThroughFiveYearsFairValue_019d02fd-4d50-7101-a5fd-377a656cc1b4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_019d02fd-4d50-73ed-b028-03c708861f1c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019d02fd-4d50-7702-8c9b-183b56726d8b" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_019d02fd-4d50-73ed-b028-03c708861f1c" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/PropertyandEquipmentNetScheduleofPropertyandEquipmentDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#PropertyandEquipmentNetScheduleofPropertyandEquipmentDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/PropertyandEquipmentNetScheduleofPropertyandEquipmentDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentLineItems_019d02fd-4d50-7745-9a4e-0cbee4751427" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_019d02fd-4d50-7f00-9961-1ec68cfb588f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_019d02fd-4d50-7745-9a4e-0cbee4751427" xlink:to="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_019d02fd-4d50-7f00-9961-1ec68cfb588f" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_019d02fd-4d50-79d1-967c-4021454ba14f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_019d02fd-4d50-7f00-9961-1ec68cfb588f" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_019d02fd-4d50-79d1-967c-4021454ba14f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_019d02fd-4d50-79d1-967c-4021454ba14f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_019d02fd-4d50-79d1-967c-4021454ba14f" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_019d02fd-4d50-79d1-967c-4021454ba14f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_019d02fd-4d50-7dd8-b32b-fb383d09c9c8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_019d02fd-4d50-79d1-967c-4021454ba14f" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_019d02fd-4d50-7dd8-b32b-fb383d09c9c8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_PropertyPlantandEquipmentExcludingConstructionInProgressMember_019d02fd-4d50-7d76-9dcc-655ac26489cc" xlink:href="docu-20260430.xsd#docu_PropertyPlantandEquipmentExcludingConstructionInProgressMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_019d02fd-4d50-7dd8-b32b-fb383d09c9c8" xlink:to="loc_docu_PropertyPlantandEquipmentExcludingConstructionInProgressMember_019d02fd-4d50-7d76-9dcc-655ac26489cc" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComputerEquipmentMember_019d02fd-4d50-771e-aeb2-a34840329c5f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ComputerEquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_docu_PropertyPlantandEquipmentExcludingConstructionInProgressMember_019d02fd-4d50-7d76-9dcc-655ac26489cc" xlink:to="loc_us-gaap_ComputerEquipmentMember_019d02fd-4d50-771e-aeb2-a34840329c5f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SoftwareAndSoftwareDevelopmentCostsMember_019d02fd-4d50-7250-8555-616be1494bfe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SoftwareAndSoftwareDevelopmentCostsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_docu_PropertyPlantandEquipmentExcludingConstructionInProgressMember_019d02fd-4d50-7d76-9dcc-655ac26489cc" xlink:to="loc_us-gaap_SoftwareAndSoftwareDevelopmentCostsMember_019d02fd-4d50-7250-8555-616be1494bfe" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FurnitureAndFixturesMember_019d02fd-4d50-7bb8-aa1a-f5e332a65056" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FurnitureAndFixturesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_docu_PropertyPlantandEquipmentExcludingConstructionInProgressMember_019d02fd-4d50-7d76-9dcc-655ac26489cc" xlink:to="loc_us-gaap_FurnitureAndFixturesMember_019d02fd-4d50-7bb8-aa1a-f5e332a65056" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LeaseholdImprovementsMember_019d02fd-4d50-7a15-a379-70f5435c133c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LeaseholdImprovementsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_docu_PropertyPlantandEquipmentExcludingConstructionInProgressMember_019d02fd-4d50-7d76-9dcc-655ac26489cc" xlink:to="loc_us-gaap_LeaseholdImprovementsMember_019d02fd-4d50-7a15-a379-70f5435c133c" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConstructionInProgressMember_019d02fd-4d50-7081-b658-3ee784534c8f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConstructionInProgressMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_019d02fd-4d50-7dd8-b32b-fb383d09c9c8" xlink:to="loc_us-gaap_ConstructionInProgressMember_019d02fd-4d50-7081-b658-3ee784534c8f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentGross_019d02fd-4d50-7d4e-abc0-8e980fb35ba9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_019d02fd-4d50-7745-9a4e-0cbee4751427" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentGross_019d02fd-4d50-7d4e-abc0-8e980fb35ba9" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_019d02fd-4d50-7d65-8193-94e9c3c09b0b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_019d02fd-4d50-7745-9a4e-0cbee4751427" xlink:to="loc_us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_019d02fd-4d50-7d65-8193-94e9c3c09b0b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentNet_019d02fd-4d50-71ef-9083-985cc0d721af" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_019d02fd-4d50-7745-9a4e-0cbee4751427" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentNet_019d02fd-4d50-71ef-9083-985cc0d721af" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/DeferredContractAcquisitionandFulfillmentCostsDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#DeferredContractAcquisitionandFulfillmentCostsDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/DeferredContractAcquisitionandFulfillmentCostsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostLineItems_019d02fd-4d50-7f1e-86d0-16255952214a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostTable_019d02fd-4d50-783e-961a-0f7dc8fd0f73" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_019d02fd-4d50-7f1e-86d0-16255952214a" xlink:to="loc_us-gaap_CapitalizedContractCostTable_019d02fd-4d50-783e-961a-0f7dc8fd0f73" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostAxis_019d02fd-4d50-7b4c-ad96-0acaf4a8befe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_CapitalizedContractCostTable_019d02fd-4d50-783e-961a-0f7dc8fd0f73" xlink:to="loc_us-gaap_CapitalizedContractCostAxis_019d02fd-4d50-7b4c-ad96-0acaf4a8befe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostDomain_019d02fd-4d50-7b4c-ad96-0acaf4a8befe_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_CapitalizedContractCostAxis_019d02fd-4d50-7b4c-ad96-0acaf4a8befe" xlink:to="loc_us-gaap_CapitalizedContractCostDomain_019d02fd-4d50-7b4c-ad96-0acaf4a8befe_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostDomain_019d02fd-4d50-7c06-82b4-d5b9687048ef" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_CapitalizedContractCostAxis_019d02fd-4d50-7b4c-ad96-0acaf4a8befe" xlink:to="loc_us-gaap_CapitalizedContractCostDomain_019d02fd-4d50-7c06-82b4-d5b9687048ef" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_ContractAcquisitionCostsMember_019d02fd-4d50-7b35-a8a5-6e69e420f478" xlink:href="docu-20260430.xsd#docu_ContractAcquisitionCostsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostDomain_019d02fd-4d50-7c06-82b4-d5b9687048ef" xlink:to="loc_docu_ContractAcquisitionCostsMember_019d02fd-4d50-7b35-a8a5-6e69e420f478" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_ContractFulfillmentCostsMember_019d02fd-4d50-750a-a842-8488ff7b5b28" xlink:href="docu-20260430.xsd#docu_ContractFulfillmentCostsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostDomain_019d02fd-4d50-7c06-82b4-d5b9687048ef" xlink:to="loc_docu_ContractFulfillmentCostsMember_019d02fd-4d50-750a-a842-8488ff7b5b28" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_CapitalizedContractCostNetRollForward_019d02fd-4d50-78a8-8fcc-427776c79cbb" xlink:href="docu-20260430.xsd#docu_CapitalizedContractCostNetRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_019d02fd-4d50-7f1e-86d0-16255952214a" xlink:to="loc_docu_CapitalizedContractCostNetRollForward_019d02fd-4d50-78a8-8fcc-427776c79cbb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostNet_019d02fd-4d50-7e2d-8611-d6d944ef21c9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_docu_CapitalizedContractCostNetRollForward_019d02fd-4d50-78a8-8fcc-427776c79cbb" xlink:to="loc_us-gaap_CapitalizedContractCostNet_019d02fd-4d50-7e2d-8611-d6d944ef21c9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_CapitalizedContractCostAdditions_019d02fd-4d50-7d1c-98f5-2a5dda695094" xlink:href="docu-20260430.xsd#docu_CapitalizedContractCostAdditions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_docu_CapitalizedContractCostNetRollForward_019d02fd-4d50-78a8-8fcc-427776c79cbb" xlink:to="loc_docu_CapitalizedContractCostAdditions_019d02fd-4d50-7d1c-98f5-2a5dda695094" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostAmortization_019d02fd-4d50-7ca3-a271-f0ef0374307f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostAmortization"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_docu_CapitalizedContractCostNetRollForward_019d02fd-4d50-78a8-8fcc-427776c79cbb" xlink:to="loc_us-gaap_CapitalizedContractCostAmortization_019d02fd-4d50-7ca3-a271-f0ef0374307f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_CapitalizedContractCostCumulativeTranslationAdjustmentIncreaseDecrease_019d02fd-4d50-7b1f-8efe-4d14c5b9e9d7" xlink:href="docu-20260430.xsd#docu_CapitalizedContractCostCumulativeTranslationAdjustmentIncreaseDecrease"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_docu_CapitalizedContractCostNetRollForward_019d02fd-4d50-78a8-8fcc-427776c79cbb" xlink:to="loc_docu_CapitalizedContractCostCumulativeTranslationAdjustmentIncreaseDecrease_019d02fd-4d50-7b1f-8efe-4d14c5b9e9d7" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostNet_019d02fd-4d50-7270-9361-be6f88840093" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostNet"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/DebtDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#DebtDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/DebtDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentLineItems_019e1d19-15c6-7f77-80be-3988d02af6a8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTable_019d02fd-4d50-730d-aa45-8505826c484b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DebtInstrumentLineItems_019e1d19-15c6-7f77-80be-3988d02af6a8" xlink:to="loc_us-gaap_DebtInstrumentTable_019d02fd-4d50-730d-aa45-8505826c484b" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityAxis_019e1d19-15c6-7f04-ab2f-02263c68bfe1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CreditFacilityAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_019d02fd-4d50-730d-aa45-8505826c484b" xlink:to="loc_us-gaap_CreditFacilityAxis_019e1d19-15c6-7f04-ab2f-02263c68bfe1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_019e1d19-15c6-7f04-ab2f-02263c68bfe1_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_CreditFacilityAxis_019e1d19-15c6-7f04-ab2f-02263c68bfe1" xlink:to="loc_us-gaap_CreditFacilityDomain_019e1d19-15c6-7f04-ab2f-02263c68bfe1_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_019e1d19-15c6-7b1b-bbdd-52d8265cfeaf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_CreditFacilityAxis_019e1d19-15c6-7f04-ab2f-02263c68bfe1" xlink:to="loc_us-gaap_CreditFacilityDomain_019e1d19-15c6-7b1b-bbdd-52d8265cfeaf" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevolvingCreditFacilityMember_019e1d19-15c6-7a11-801e-8c563ea5d20b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevolvingCreditFacilityMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CreditFacilityDomain_019e1d19-15c6-7b1b-bbdd-52d8265cfeaf" xlink:to="loc_us-gaap_RevolvingCreditFacilityMember_019e1d19-15c6-7a11-801e-8c563ea5d20b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_019e1d19-15c6-7988-a5da-bc4d427d4759" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_019d02fd-4d50-730d-aa45-8505826c484b" xlink:to="loc_us-gaap_DebtInstrumentAxis_019e1d19-15c6-7988-a5da-bc4d427d4759" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_019e1d19-15c6-7988-a5da-bc4d427d4759_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_019e1d19-15c6-7988-a5da-bc4d427d4759" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_019e1d19-15c6-7988-a5da-bc4d427d4759_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_019e1d19-15c6-723a-9127-0cc1b26c8122" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_019e1d19-15c6-7988-a5da-bc4d427d4759" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_019e1d19-15c6-723a-9127-0cc1b26c8122" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_May2025CreditAgreementMember_019e1d19-15c6-725c-9b69-1f2ed41e9f3f" xlink:href="docu-20260430.xsd#docu_May2025CreditAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_019e1d19-15c6-723a-9127-0cc1b26c8122" xlink:to="loc_docu_May2025CreditAgreementMember_019e1d19-15c6-725c-9b69-1f2ed41e9f3f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_019e1d19-15c6-7dc0-b9cc-7350753fecf9" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_019d02fd-4d50-730d-aa45-8505826c484b" xlink:to="loc_srt_RangeAxis_019e1d19-15c6-7dc0-b9cc-7350753fecf9" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_019e1d19-15c6-7dc0-b9cc-7350753fecf9_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_019e1d19-15c6-7dc0-b9cc-7350753fecf9" xlink:to="loc_srt_RangeMember_019e1d19-15c6-7dc0-b9cc-7350753fecf9_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_019e1d19-15c6-732a-a1d9-c3751fd68ad9" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_019e1d19-15c6-7dc0-b9cc-7350753fecf9" xlink:to="loc_srt_RangeMember_019e1d19-15c6-732a-a1d9-c3751fd68ad9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_019e1d19-15c6-7b6a-b8af-44e084a4dd50" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_019e1d19-15c6-732a-a1d9-c3751fd68ad9" xlink:to="loc_srt_MinimumMember_019e1d19-15c6-7b6a-b8af-44e084a4dd50" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MaximumMember_019e1d19-15c6-72c9-87c6-eaa5c7a785e3" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_019e1d19-15c6-732a-a1d9-c3751fd68ad9" xlink:to="loc_srt_MaximumMember_019e1d19-15c6-72c9-87c6-eaa5c7a785e3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity_019e1d19-15c6-78b8-9740-a2e71b05e868" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_019e1d19-15c6-7f77-80be-3988d02af6a8" xlink:to="loc_us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity_019e1d19-15c6-78b8-9740-a2e71b05e868" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_LineOfCreditFacilityAccordionFeatureIncreaseLimit_019e1d19-15c6-7dc0-8c5d-54083507485a" xlink:href="docu-20260430.xsd#docu_LineOfCreditFacilityAccordionFeatureIncreaseLimit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_019e1d19-15c6-7f77-80be-3988d02af6a8" xlink:to="loc_docu_LineOfCreditFacilityAccordionFeatureIncreaseLimit_019e1d19-15c6-7dc0-8c5d-54083507485a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebt_019e1d19-15c6-77d6-b67b-1854c883689a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongTermDebt"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_019e1d19-15c6-7f77-80be-3988d02af6a8" xlink:to="loc_us-gaap_LongTermDebt_019e1d19-15c6-77d6-b67b-1854c883689a" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityUnusedCapacityCommitmentFeePercentage_019e1d19-15c6-7e7d-8451-659f5931323f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditFacilityUnusedCapacityCommitmentFeePercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_019e1d19-15c6-7f77-80be-3988d02af6a8" xlink:to="loc_us-gaap_LineOfCreditFacilityUnusedCapacityCommitmentFeePercentage_019e1d19-15c6-7e7d-8451-659f5931323f" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/CommitmentsandContingenciesNarrativeDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#CommitmentsandContingenciesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/CommitmentsandContingenciesNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherCommitmentsLineItems_019d02fd-4d50-7095-8012-db0a669c540b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherCommitmentsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherCommitmentsTable_019d02fd-4d50-7350-bd86-69e700e1a49d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherCommitmentsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_OtherCommitmentsLineItems_019d02fd-4d50-7095-8012-db0a669c540b" xlink:to="loc_us-gaap_OtherCommitmentsTable_019d02fd-4d50-7350-bd86-69e700e1a49d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis_019d02fd-4d50-7fe0-aa5d-7ee4d772da73" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_OtherCommitmentsTable_019d02fd-4d50-7350-bd86-69e700e1a49d" xlink:to="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis_019d02fd-4d50-7fe0-aa5d-7ee4d772da73" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_019d02fd-4d50-7fe0-aa5d-7ee4d772da73_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis_019d02fd-4d50-7fe0-aa5d-7ee4d772da73" xlink:to="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_019d02fd-4d50-7fe0-aa5d-7ee4d772da73_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_019d02fd-4d50-7f0d-a619-e3e228ca0cc4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis_019d02fd-4d50-7fe0-aa5d-7ee4d772da73" xlink:to="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_019d02fd-4d50-7f0d-a619-e3e228ca0cc4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_CloudComputingServiceProviderAgreementThroughFiscal2028Member_019d02fd-4d50-7d4c-b4f4-770dc8bf6475" xlink:href="docu-20260430.xsd#docu_CloudComputingServiceProviderAgreementThroughFiscal2028Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_019d02fd-4d50-7f0d-a619-e3e228ca0cc4" xlink:to="loc_docu_CloudComputingServiceProviderAgreementThroughFiscal2028Member_019d02fd-4d50-7d4c-b4f4-770dc8bf6475" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationBalanceSheetAmount_019d02fd-4d50-779e-8e13-4fa426ccb1a9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnrecordedUnconditionalPurchaseObligationBalanceSheetAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OtherCommitmentsLineItems_019d02fd-4d50-7095-8012-db0a669c540b" xlink:to="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationBalanceSheetAmount_019d02fd-4d50-779e-8e13-4fa426ccb1a9" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_NumberOfPutativeShareholderDerivativeCasesFiledToDate_019d02fd-4d50-7abd-beba-309a600410e9" xlink:href="docu-20260430.xsd#docu_NumberOfPutativeShareholderDerivativeCasesFiledToDate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OtherCommitmentsLineItems_019d02fd-4d50-7095-8012-db0a669c540b" xlink:to="loc_docu_NumberOfPutativeShareholderDerivativeCasesFiledToDate_019d02fd-4d50-7abd-beba-309a600410e9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNewClaimsFiledNumber_019d02fd-4d50-7195-8b81-49d8e92919ef" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingencyNewClaimsFiledNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OtherCommitmentsLineItems_019d02fd-4d50-7095-8012-db0a669c540b" xlink:to="loc_us-gaap_LossContingencyNewClaimsFiledNumber_019d02fd-4d50-7195-8b81-49d8e92919ef" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/StockholdersEquityNarrativeDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#StockholdersEquityNarrativeDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/StockholdersEquityNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019d02fd-4d50-7251-b583-2990a1a7365c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019d02fd-4d50-7251-b583-2990a1a7365c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_019d02fd-4d50-7ad4-9050-ec55eccac4ca" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019d02fd-4d50-7251-b583-2990a1a7365c" xlink:to="loc_us-gaap_PlanNameAxis_019d02fd-4d50-7ad4-9050-ec55eccac4ca" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019d02fd-4d50-7ad4-9050-ec55eccac4ca_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_019d02fd-4d50-7ad4-9050-ec55eccac4ca" xlink:to="loc_us-gaap_PlanNameDomain_019d02fd-4d50-7ad4-9050-ec55eccac4ca_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019d02fd-4d50-71e9-bc6d-f7326073e871" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_019d02fd-4d50-7ad4-9050-ec55eccac4ca" xlink:to="loc_us-gaap_PlanNameDomain_019d02fd-4d50-71e9-bc6d-f7326073e871" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_A2018EquityIncentivePlanMember_019d02fd-4d50-775b-98e8-3072ec26ecef" xlink:href="docu-20260430.xsd#docu_A2018EquityIncentivePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_019d02fd-4d50-71e9-bc6d-f7326073e871" xlink:to="loc_docu_A2018EquityIncentivePlanMember_019d02fd-4d50-775b-98e8-3072ec26ecef" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_EmployeeStockPurchasePlanMember_019d02fd-4d50-75bd-b5ed-cbe443688968" xlink:href="docu-20260430.xsd#docu_EmployeeStockPurchasePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_019d02fd-4d50-71e9-bc6d-f7326073e871" xlink:to="loc_docu_EmployeeStockPurchasePlanMember_019d02fd-4d50-75bd-b5ed-cbe443688968" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019d02fd-4d50-7112-924b-514d0cebb8fd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019d02fd-4d50-7251-b583-2990a1a7365c" xlink:to="loc_us-gaap_AwardTypeAxis_019d02fd-4d50-7112-924b-514d0cebb8fd" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-7112-924b-514d0cebb8fd_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019d02fd-4d50-7112-924b-514d0cebb8fd" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-7112-924b-514d0cebb8fd_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-705c-8a55-6c8773ef0ce6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019d02fd-4d50-7112-924b-514d0cebb8fd" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-705c-8a55-6c8773ef0ce6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019d02fd-4d50-7e60-9c29-6d3871a817e7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-705c-8a55-6c8773ef0ce6" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019d02fd-4d50-7e60-9c29-6d3871a817e7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_019d02fd-4d50-718d-be4f-a121d46e45db" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-705c-8a55-6c8773ef0ce6" xlink:to="loc_us-gaap_EmployeeStockMember_019d02fd-4d50-718d-be4f-a121d46e45db" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_VestingAxis_019d02fd-4d50-7aba-a583-6215e20368c2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019d02fd-4d50-7251-b583-2990a1a7365c" xlink:to="loc_us-gaap_VestingAxis_019d02fd-4d50-7aba-a583-6215e20368c2" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_VestingDomain_019d02fd-4d50-7aba-a583-6215e20368c2_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_VestingAxis_019d02fd-4d50-7aba-a583-6215e20368c2" xlink:to="loc_us-gaap_VestingDomain_019d02fd-4d50-7aba-a583-6215e20368c2_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_VestingDomain_019d02fd-4d50-7e6a-98cf-73c994c718d6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_VestingAxis_019d02fd-4d50-7aba-a583-6215e20368c2" xlink:to="loc_us-gaap_VestingDomain_019d02fd-4d50-7e6a-98cf-73c994c718d6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_MarketBasedAndPerformanceBasedVestingConditionsMember_019d02fd-4d50-7b65-8728-97934f00b52a" xlink:href="docu-20260430.xsd#docu_MarketBasedAndPerformanceBasedVestingConditionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_VestingDomain_019d02fd-4d50-7e6a-98cf-73c994c718d6" xlink:to="loc_docu_MarketBasedAndPerformanceBasedVestingConditionsMember_019d02fd-4d50-7b65-8728-97934f00b52a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfPlans_019d02fd-4d50-72c7-a0a0-5c8a464f3063" xlink:href="docu-20260430.xsd#docu_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfPlans"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_docu_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfPlans_019d02fd-4d50-72c7-a0a0-5c8a464f3063" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_019d02fd-4d50-7344-8d6f-01fe0f814858" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_019d02fd-4d50-7344-8d6f-01fe0f814858" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions_019d02fd-4d50-7eed-994a-da7ab98dcb8d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions_019d02fd-4d50-7eed-994a-da7ab98dcb8d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_019d02fd-4d50-7e64-bf4d-0ee056a97bc1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_019d02fd-4d50-7e64-bf4d-0ee056a97bc1" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueNonvested_019d02fd-4d50-7bfd-ab25-6f26b1e139cd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueNonvested"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueNonvested_019d02fd-4d50-7bfd-ab25-6f26b1e139cd" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodPerformanceTargetPercentage_019d02fd-4d50-7763-bc8e-a8705f55b9ed" xlink:href="docu-20260430.xsd#docu_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodPerformanceTargetPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_docu_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodPerformanceTargetPercentage_019d02fd-4d50-7763-bc8e-a8705f55b9ed" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_019d02fd-4d50-7c0b-a24a-b87e972df0de" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_019d02fd-4d50-7c0b-a24a-b87e972df0de" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_StockRepurchaseProgramAdditionalAuthorizedAmount_019e2124-7765-7886-a805-f5c303b526dc" xlink:href="docu-20260430.xsd#docu_StockRepurchaseProgramAdditionalAuthorizedAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_docu_StockRepurchaseProgramAdditionalAuthorizedAmount_019e2124-7765-7886-a805-f5c303b526dc" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StockRepurchaseProgramAuthorizedAmount1_019d02fd-4d50-7924-85f7-797430d79645" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StockRepurchaseProgramAuthorizedAmount1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_srt_StockRepurchaseProgramAuthorizedAmount1_019d02fd-4d50-7924-85f7-797430d79645" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchaseProgramRemainingAuthorizedRepurchaseAmount1_019d02fd-4d50-7cec-8a29-ed4bc80e9621" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockRepurchaseProgramRemainingAuthorizedRepurchaseAmount1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7e0e-9f6f-d64625c89259" xlink:to="loc_us-gaap_StockRepurchaseProgramRemainingAuthorizedRepurchaseAmount1_019d02fd-4d50-7cec-8a29-ed4bc80e9621" xlink:type="arc" order="9"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/StockholdersEquityScheduleofRSUActivityDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#StockholdersEquityScheduleofRSUActivityDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/StockholdersEquityScheduleofRSUActivityDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7745-8452-2158b99ed280" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019d02fd-4d50-7928-9042-a89699a8d781" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7745-8452-2158b99ed280" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019d02fd-4d50-7928-9042-a89699a8d781" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019d02fd-4d50-7a45-b348-ed3127adb199" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019d02fd-4d50-7928-9042-a89699a8d781" xlink:to="loc_us-gaap_AwardTypeAxis_019d02fd-4d50-7a45-b348-ed3127adb199" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-7a45-b348-ed3127adb199_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019d02fd-4d50-7a45-b348-ed3127adb199" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-7a45-b348-ed3127adb199_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-7c0c-aff3-ace41d9ed476" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019d02fd-4d50-7a45-b348-ed3127adb199" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-7c0c-aff3-ace41d9ed476" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019d02fd-4d50-7d1c-a7a0-6843991eb7bd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019d02fd-4d50-7c0c-aff3-ace41d9ed476" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019d02fd-4d50-7d1c-a7a0-6843991eb7bd" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019d02fd-4d50-726d-a46b-f1ea1f88ed24" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7745-8452-2158b99ed280" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019d02fd-4d50-726d-a46b-f1ea1f88ed24" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_019d02fd-4d50-7311-b360-a621264bad7e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019d02fd-4d50-726d-a46b-f1ea1f88ed24" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_019d02fd-4d50-7311-b360-a621264bad7e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_019d02fd-4d50-71f9-a18f-ad97823f4fdf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019d02fd-4d50-726d-a46b-f1ea1f88ed24" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_019d02fd-4d50-71f9-a18f-ad97823f4fdf" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod_019d02fd-4d50-793e-b5a9-ab06bda819a1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019d02fd-4d50-726d-a46b-f1ea1f88ed24" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod_019d02fd-4d50-793e-b5a9-ab06bda819a1" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod_019d02fd-4d50-7c8f-8492-8b35da29cad4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019d02fd-4d50-726d-a46b-f1ea1f88ed24" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod_019d02fd-4d50-7c8f-8492-8b35da29cad4" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_019d02fd-4d50-798e-9955-d0b8fa37807e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019d02fd-4d50-71cf-b7b8-3cdb54f8cf4c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019d02fd-4d50-7745-8452-2158b99ed280" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019d02fd-4d50-71cf-b7b8-3cdb54f8cf4c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_019d02fd-4d50-7e5d-b9ba-0c331e4aedb2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019d02fd-4d50-71cf-b7b8-3cdb54f8cf4c" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_019d02fd-4d50-7e5d-b9ba-0c331e4aedb2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_019d02fd-4d50-71aa-bab6-ccce84ae5d0c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019d02fd-4d50-71cf-b7b8-3cdb54f8cf4c" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_019d02fd-4d50-71aa-bab6-ccce84ae5d0c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue_019d02fd-4d50-7373-9197-eae5dc0afcef" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019d02fd-4d50-71cf-b7b8-3cdb54f8cf4c" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue_019d02fd-4d50-7373-9197-eae5dc0afcef" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue_019d02fd-4d50-7d5e-9f98-21980f2faba9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019d02fd-4d50-71cf-b7b8-3cdb54f8cf4c" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue_019d02fd-4d50-7d5e-9f98-21980f2faba9" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_019d02fd-4d50-78fd-b21a-8e5fdbd4edcb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/NetIncomeperShareAttributabletoCommonStockholdersScheduleofAntidilutiveSecuritiesDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#NetIncomeperShareAttributabletoCommonStockholdersScheduleofAntidilutiveSecuritiesDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/NetIncomeperShareAttributabletoCommonStockholdersScheduleofAntidilutiveSecuritiesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019d02fd-4d50-78fc-8d92-16aec67bcc2d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019d02fd-4d50-7a9f-8d5b-3028c6d277a4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019d02fd-4d50-78fc-8d92-16aec67bcc2d" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019d02fd-4d50-7a9f-8d5b-3028c6d277a4" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019d02fd-4d50-73ed-a724-a44944a8073a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019d02fd-4d50-7a9f-8d5b-3028c6d277a4" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019d02fd-4d50-73ed-a724-a44944a8073a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019d02fd-4d50-73ed-a724-a44944a8073a_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019d02fd-4d50-73ed-a724-a44944a8073a" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019d02fd-4d50-73ed-a724-a44944a8073a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019d02fd-4d50-716c-870b-98c0a4e5533a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019d02fd-4d50-73ed-a724-a44944a8073a" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019d02fd-4d50-716c-870b-98c0a4e5533a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019d02fd-4d50-7b7a-ae28-8f0c4459549d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019d02fd-4d50-716c-870b-98c0a4e5533a" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019d02fd-4d50-7b7a-ae28-8f0c4459549d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_019d02fd-4d50-7eee-9290-690051f63055" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019d02fd-4d50-716c-870b-98c0a4e5533a" xlink:to="loc_us-gaap_EmployeeStockMember_019d02fd-4d50-7eee-9290-690051f63055" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_019d02fd-4d50-7155-ad8f-ee86e849fe77" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019d02fd-4d50-78fc-8d92-16aec67bcc2d" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_019d02fd-4d50-7155-ad8f-ee86e849fe77" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/SegmentandGeographicInformationScheduleofSegmentProfitorLossDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#SegmentandGeographicInformationScheduleofSegmentProfitorLossDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/SegmentandGeographicInformationScheduleofSegmentProfitorLossDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentReportingInformationLineItems_019d02fd-4d50-76f4-80f3-eb5f461fe0d1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentReportingInformationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019d02fd-4d50-7269-a1af-79141e1ad833" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019d02fd-4d50-76f4-80f3-eb5f461fe0d1" xlink:to="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019d02fd-4d50-7269-a1af-79141e1ad833" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementBusinessSegmentsAxis_019d02fd-4d50-79e8-b9fd-0614201e94f6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementBusinessSegmentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019d02fd-4d50-7269-a1af-79141e1ad833" xlink:to="loc_us-gaap_StatementBusinessSegmentsAxis_019d02fd-4d50-79e8-b9fd-0614201e94f6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_019d02fd-4d50-79e8-b9fd-0614201e94f6_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_019d02fd-4d50-79e8-b9fd-0614201e94f6" xlink:to="loc_us-gaap_SegmentDomain_019d02fd-4d50-79e8-b9fd-0614201e94f6_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_019d02fd-4d50-7ec5-8669-fa4c2da0383d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_019d02fd-4d50-79e8-b9fd-0614201e94f6" xlink:to="loc_us-gaap_SegmentDomain_019d02fd-4d50-7ec5-8669-fa4c2da0383d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_ReportableSegmentMember_019d02fd-4d50-75bc-941a-3b66ded1e6db" xlink:href="docu-20260430.xsd#docu_ReportableSegmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentDomain_019d02fd-4d50-7ec5-8669-fa4c2da0383d" xlink:to="loc_docu_ReportableSegmentMember_019d02fd-4d50-75bc-941a-3b66ded1e6db" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_019d02fd-4d50-798c-b7e0-eb80fddb40e3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019d02fd-4d50-76f4-80f3-eb5f461fe0d1" xlink:to="loc_us-gaap_NetIncomeLoss_019d02fd-4d50-798c-b7e0-eb80fddb40e3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019d02fd-4d50-72cf-80fe-e7001b6fd330" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019d02fd-4d50-76f4-80f3-eb5f461fe0d1" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019d02fd-4d50-72cf-80fe-e7001b6fd330" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DepreciationDepletionAndAmortization_019d02fd-4d50-7f07-ba0e-ebcf7c30e08a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DepreciationDepletionAndAmortization"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019d02fd-4d50-76f4-80f3-eb5f461fe0d1" xlink:to="loc_us-gaap_DepreciationDepletionAndAmortization_019d02fd-4d50-7f07-ba0e-ebcf7c30e08a" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InvestmentIncomeInterest_019d02fd-4d50-79d7-b23c-dc2469faad58" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InvestmentIncomeInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019d02fd-4d50-76f4-80f3-eb5f461fe0d1" xlink:to="loc_us-gaap_InvestmentIncomeInterest_019d02fd-4d50-79d7-b23c-dc2469faad58" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeTaxExpenseBenefit_019d02fd-4d50-7ada-8176-7e3f22c56d44" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxExpenseBenefit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019d02fd-4d50-76f4-80f3-eb5f461fe0d1" xlink:to="loc_us-gaap_IncomeTaxExpenseBenefit_019d02fd-4d50-7ada-8176-7e3f22c56d44" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/SegmentandGeographicInformationScheduleofRevenuesbyGeographicAreaDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#SegmentandGeographicInformationScheduleofRevenuesbyGeographicAreaDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/SegmentandGeographicInformationScheduleofRevenuesbyGeographicAreaDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems_019d02fd-4d50-7155-b679-36a834622001" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable_019d02fd-4d50-7a1a-9f43-0551e53ad57a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems_019d02fd-4d50-7155-b679-36a834622001" xlink:to="loc_us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable_019d02fd-4d50-7a1a-9f43-0551e53ad57a" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementGeographicalAxis_019d02fd-4d50-7bff-9353-ed937bbe6c66" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable_019d02fd-4d50-7a1a-9f43-0551e53ad57a" xlink:to="loc_srt_StatementGeographicalAxis_019d02fd-4d50-7bff-9353-ed937bbe6c66" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_019d02fd-4d50-7bff-9353-ed937bbe6c66_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementGeographicalAxis_019d02fd-4d50-7bff-9353-ed937bbe6c66" xlink:to="loc_srt_SegmentGeographicalDomain_019d02fd-4d50-7bff-9353-ed937bbe6c66_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_019d02fd-4d50-7f69-b18e-ac96e847b2d2" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementGeographicalAxis_019d02fd-4d50-7bff-9353-ed937bbe6c66" xlink:to="loc_srt_SegmentGeographicalDomain_019d02fd-4d50-7f69-b18e-ac96e847b2d2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_country_US_019d02fd-4d50-7d87-a5cb-9cabeda9bf85" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_US"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_019d02fd-4d50-7f69-b18e-ac96e847b2d2" xlink:to="loc_country_US_019d02fd-4d50-7d87-a5cb-9cabeda9bf85" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NonUsMember_019d02fd-4d50-76ce-8adb-578738e727f2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NonUsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_019d02fd-4d50-7f69-b18e-ac96e847b2d2" xlink:to="loc_us-gaap_NonUsMember_019d02fd-4d50-76ce-8adb-578738e727f2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019d02fd-4d50-78b0-90a9-355fe683b20a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems_019d02fd-4d50-7155-b679-36a834622001" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019d02fd-4d50-78b0-90a9-355fe683b20a" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://docusign.com/role/SubsequentEventsDetails" xlink:type="simple" xlink:href="docu-20260430.xsd#SubsequentEventsDetails"/>
  <link:definitionLink xlink:role="http://docusign.com/role/SubsequentEventsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventLineItems_019e2136-c0b1-7d2f-a6b7-8729870c164b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTable_019d02fd-4d50-7e5e-bd3c-e0abce952b7a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SubsequentEventLineItems_019e2136-c0b1-7d2f-a6b7-8729870c164b" xlink:to="loc_us-gaap_SubsequentEventTable_019d02fd-4d50-7e5e-bd3c-e0abce952b7a" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeAxis_019e2136-c0b1-796a-a996-17d7742a387d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_019d02fd-4d50-7e5e-bd3c-e0abce952b7a" xlink:to="loc_us-gaap_SubsequentEventTypeAxis_019e2136-c0b1-796a-a996-17d7742a387d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_019e2136-c0b1-796a-a996-17d7742a387d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_019e2136-c0b1-796a-a996-17d7742a387d" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_019e2136-c0b1-796a-a996-17d7742a387d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_019e2136-c0b1-7aad-bb67-29eebfaf46a4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_019e2136-c0b1-796a-a996-17d7742a387d" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_019e2136-c0b1-7aad-bb67-29eebfaf46a4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventMember_019e2136-c0b1-7db0-835c-efc670a29c45" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventTypeDomain_019e2136-c0b1-7aad-bb67-29eebfaf46a4" xlink:to="loc_us-gaap_SubsequentEventMember_019e2136-c0b1-7db0-835c-efc670a29c45" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue_019e6488-3e94-7778-9bdf-fc98b37a906f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_019e2136-c0b1-7d2f-a6b7-8729870c164b" xlink:to="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue_019e6488-3e94-7778-9bdf-fc98b37a906f" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/IndividualsOnly" xlink:type="simple" xlink:href="https://xbrl.sec.gov/ecd/2025/ecd-2025.xsd#IndividualsOnly"/>
  <link:definitionLink xlink:role="http://xbrl.sec.gov/ecd/role/IndividualsOnly" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_ecd_AllIndividualsMember" xlink:href="https://xbrl.sec.gov/ecd/2025/ecd-2025.xsd#ecd_AllIndividualsMember"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_RobertChatwaniMember_019e6493-2ed3-7377-a81f-fabaf9feea8b" xlink:href="docu-20260430.xsd#docu_RobertChatwaniMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_ecd_AllIndividualsMember" xlink:to="loc_docu_RobertChatwaniMember_019e6493-2ed3-7377-a81f-fabaf9feea8b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_docu_BlakeGraysonMember_019d02fd-4bb0-7cc7-85aa-c103a865af88" xlink:href="docu-20260430.xsd#docu_BlakeGraysonMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_ecd_AllIndividualsMember" xlink:to="loc_docu_BlakeGraysonMember_019d02fd-4bb0-7cc7-85aa-c103a865af88" xlink:type="arc" order="3"/>
  </link:definitionLink>
</link:linkbase>
