<p>The COVID-19 pandemic has caused significant global health and economic disruption. Mutations E166N, E166R, E166N, S144A and His163A in the SARS-CoV-2 main protease (M^pro) have been implicated in reducing the efficacy of certain antiviral treatments. Bofutrelvir, a promising inhibitor, has shown effectiveness against SARS-CoV-2 M^pro. This study aims to evaluate the inhibitory effects of Bofutrelvir on the E166N, E166R, His163A, E166V and S144A mutants of SARS-CoV-2 M^pro, as well as on MERS-CoV M^pro. Our findings indicate a substantial reduction in the inhibitory potency of Bofutrelvir against these mutants and MERS-CoV, with IC₅₀ values significantly higher than those for the wild-type SARS-CoV-2 M^pro. Specifically, the E166N, E166R, E166V, S144A, and H163A mutations significantly reduce the binding affinity and inhibitory effectiveness of Bofutrelvir due to disrupted hydrogen bonds, altered binding site stability, and reduced enzyme activity. Structural analysis of the crystal complexes showed that changes in interactions at the S1 subsite in the mutants and the loss of hydrogen bonds at the S4 subsite in MERS-CoV M^pro are critical factors contributing to the diminished inhibitory activity. These insights reveal the necessity of ongoing structural analysis to adapt therapeutic strategies.</p>