 Abstract Encitrelvia, an oral antiviral agent that targets a SARS-CoV-2 main protease, 3CL-PRO or NSP-5, is clinically useful against SARS-CoV-2, including its amicron variants. Since most amicron subvariants have reduced sensitivity to most monoclonal antibody therapies, SARS-CoV-2 resistance to other antivirals including main protease inhibitors such as Encitrelvia is a major public health concern. Here, repeating passages of SARS-CoV-2 in the presence of Encitrelvia revealed that the M49L and E166A substitutions in NSP-5 are responsible for reduced sensitivity to Encitrelvia. Both substitutions reduced in vitro virus growth in the absence of Encitrelvia. The virus possessing NSP-5, M49L showed similar pathogenicity to wild-type virus, whereas the virus possessing NSP-5, E166A, or NSP-5, M49L, E166A slightly attenuated. Encitrelvia treatment of hamsters infected with.