<?xml version="1.0"?>
<ownershipDocument>

    <schemaVersion>X0206</schemaVersion>

    <documentType>3</documentType>

    <periodOfReport>2025-07-08</periodOfReport>

    <noSecuritiesOwned>0</noSecuritiesOwned>

    <issuer>
        <issuerCik>0001980845</issuerCik>
        <issuerName>enGene Holdings Inc.</issuerName>
        <issuerTradingSymbol>ENGN</issuerTradingSymbol>
    </issuer>

    <reportingOwner>
        <reportingOwnerId>
            <rptOwnerCik>0001376491</rptOwnerCik>
            <rptOwnerName>Astley-Sparke Philip</rptOwnerName>
        </reportingOwnerId>
        <reportingOwnerAddress>
            <rptOwnerStreet1>C/O 4868 RUE LEVY, SUITE 220</rptOwnerStreet1>
            <rptOwnerStreet2></rptOwnerStreet2>
            <rptOwnerCity>SAINT-LAURENT</rptOwnerCity>
            <rptOwnerState>A8</rptOwnerState>
            <rptOwnerZipCode>H4R 2P1</rptOwnerZipCode>
            <rptOwnerStateDescription>QUEBEC, CANADA</rptOwnerStateDescription>
        </reportingOwnerAddress>
        <reportingOwnerRelationship>
            <isDirector>true</isDirector>
        </reportingOwnerRelationship>
    </reportingOwner>

    <nonDerivativeTable>
        <nonDerivativeHolding>
            <securityTitle>
                <value>Common Shares</value>
            </securityTitle>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>20000</value>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>D</value>
                </directOrIndirectOwnership>
            </ownershipNature>
        </nonDerivativeHolding>
    </nonDerivativeTable>

    <remarks>Exhibit 24 - Power of Attorney</remarks>

    <ownerSignature>
        <signatureName>/s/ Lee Giguere, as attorney-in-fact for the Reporting Person</signatureName>
        <signatureDate>2025-07-08</signatureDate>
    </ownerSignature>
</ownershipDocument>
