This paper focuses on the impact of the Smart Specialisation Strategy (S3) and Industry 4.0 (I4) initiatives during the 2014-2020 programming period on firms’ performance in Italy. By analysing European Regional Development Fund (ERDF)-funded projects under these frameworks, we use OpenCoesione data and a Difference-in-Differences approach to assess the effectiveness of S3 and I4 initiatives. Our results reveal that projects integrating I4 technologies within the S3 framework (S3I4 projects) significantly enhance firms’ performance. This is particularly evident when compared to projects funded under other ERDF initiatives. The study highlights the importance of aligning S3 and I4 strategies with regional economic profiles and innovation capacities to maximise their impact. Our analysis underscores the role of these initiatives in driving innovation and economic growth. The results offer key insights for policymakers, suggesting that focused and strategic investment in S3 and I4 can lead to more effective regional innovation and development.