Feline infectious peritonitis (FIP) is a highly fatal disease caused by a virulent feline coronavirus in domestic and wild cats. We have previously reported the synthesis of potent coronavirus 3C-like protease (3CLpro) inhibitors and the efficacy of a protease inhibitor, GC376, in client-owned cats with FIP. In this study, we studied the effect of the amino acid changes in 3CLpro of feline coronavirus from a feline patient who received antiviral treatment for prolonged duration. We generated recombinant 3CLpro containing the identified amino acid changes (N25S, A252S or K260 N) and determined their susceptibility to protease inhibitors in the fluorescence resonance energy transfer assay. The assay showed that N25S in 3CLpro confers a small change (up to 1.68-fold increase in the 50% inhibitory concentration) in susceptibility to GC376, but other amino acid changes do not affect susceptibility. Modelling of 3CLpro carrying the amino acid changes was conducted to probe the structural basis for these findings. The results of this study may explain the observed absence of clinical resistance to the long-term antiviral treatment in the patients.