Expertise and autonomy are cornerstones to the effective operation and legitimacy of European Regulatory Agencies (ERAs). Yet, we know little about ERAs' actual autonomy, nor about factors shaping it. This article studies ERAs' actual autonomy from public and private actors, emphasising two crucial explanatory factors: expertise and rulemaking competences. The lack of insights on expertise is particularly striking, as expertise-the "raison d'être" and main resource of expert bodies-provides ERAs with a potentially powerful means to increase autonomy. Relying on a rational institutionalist framework within which ERAs enjoy substantive discretion to pursue their goals, the study empirically compares three powerful ERAs-the European Medicines Agency, the European Chemicals Agency, and the European Food Safety Authority. Based on the analysis of 39 semi-structured expert interviews, findings show that expertise is a crucial explanation for ERAs' substantive autonomy from the Commission. Towards research intensive private stakeholders, the role of expertise becomes less pronounced. Instead, ERAs are more successful in protecting their autonomy by engaging in the risk-averse interpretation of the regulatory framework and by adapting rules over time to adapt their needs: they engage in "procedural insulation". Political salience provides a scope condition for ERAs to use expert knowledge and rulemaking competences more strategically-potentially undermining scientific quality.