The latest regulatory framework, which has been introduced globally in the form of Basel III, and its implementation in the legislation of the member states of the European Union has generated much interest in the impact of regulation on the efficiency and profitability of banks. This study aims to examine the impact of the introduction of two major regulatory changes (Basel II and Basel III) on bank performance, in terms of bank size and bank-specific and macroeconomic variables. A two-stage empirical analysis was conducted on a sample of 433 European commercial banks over the 2006–2015 period. In the first stage, relative efficiency was calculated using non-parametric data envelopment analysis. In the second stage, the generalized method of moments was used to examine the impact of bank-specific and macroeconomic variables as well as regulation on bank performance, that is, profitability and efficiency. Considering bank size, the results show a diverse impact of regulation on bank performance. Regarding large- and medium-sized banks, regulation positively affects both efficiency and profitability, whereas, for small banks, it negatively affects performance. The results suggest that larger banks have skillfully adapted to the new regulatory environment. In contrast, small banks have problems with profitability and efficiency because the new regulatory framework has imposed additional administrative and regulatory burdens. Our results strongly suggest that regulation should not be implemented equally for all banks ; that is, on a one size fits all terms. A distinction between small and large banks when introducing new regulatory frameworks should be made if a reasonable level of competition is to be preserved.