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This study contributes to studies of the nexus between descriptive and substantive representation in legislatures, with a focus on gender quotas. Prior research establishes that gender quotas increase women's descriptive representation, which in turn enhances substantive representation, especially in policy areas pertaining to welfare, health, and education. However, the dynamics of this relationship in multilevel governance systems have received limited attention. The European Parliament, embodying both supranational governance and direct policy impact across its 27 member states, serves as an exemplary model for analyzing representation in a multi-level governance system. Utilizing a difference-in-differences approach to causal inference, this research assesses the effect of gender quotas in European Parliament elections on substantive representation across various policy domains. The findings extend the current understanding of political representation, particularly shedding light on the mechanisms of substantive representation in multi-level governance systems.