The norovirus 3C-like protease is a member of the chymotrypsin-like serine protease superfamily. Previous characterization of its crystal structure has implicated the Glu54–His30–Cys139 triad in the catalysis. In the present study, the Glu54 residue of the protease was subjected to site-saturation mutagenesis, with the result that nearly half of the mutants retained the significant proteolytic activity. It was suggested that a carboxylate at position 54 was not essential for the activity. The in vitro assays of the proteolysis revealed that most of Glu54 mutants retained relatively high proteolytic activity. When the Glu54 mutation was combined with the Ser mutation of the Cys139 residue, a nucleophile, only the Asp54 and Gln54 mutations showed proteolytic activity comparable to that of the Ser139 single mutant, suggesting that a hydrogen bond between Glu54 and His30 was critical in the Ser139 background. These results suggested that the mechanism of the proteolysis by the wild-type norovirus 3C-like protease was different from that of typical chymotrypsin-like serine proteases.