Staphylococcus aureus (S. aureus) is a ubiquitous Gram-positive pathogenic bacterium responsible for a majority of skin infections and toxic shock syndromes. In this study, a 34-kDa glutamate-specific serine protease (named VSPase) secreted by a clinical isolate of S. aureus sp. strain C-66 was purified and characterized, and VSPase-encoding gene was also cloned by PCR. VSPase enzyme purified from culture supernatant and its recombinant enzyme expressed in E. coli exhibited a proteolytic activity over a broad range of pH (6.0-8.5) and showed an optimal activity at 45 ˚C. The enzyme activity was completely inhibited by DFP. The N-terminal sequence of native VSPase showed that the enzyme was produced as a form of zymogen and activated to a functional enzyme by losing its N-terminal 68 amino acid residues. VSPase specifically cleaved peptide bonds at the carboxyl sides of glutamate residues in a protein substrate such as prothrombin and exhibited its amidolytic activity towards a chromogenic substrate, Z-Phe-Leu-Glu-pNA (L-2135). The Km, kcat and kcat/Km values for VSPase were estimated to be 1.48 ± 0.156 mM, 44.4 ± 2.66/sec and 30/mM/sec, respectively, when L-2135 was used as a substrate. It was revealed by site-directed mutagenesis that one of substitution mutations resulted in His119, Asp161 and Ser237 residues of VSPase abolished the enzyme activity dramatically, suggesting that the three amino acid residues may compose a catalytic triad in VSPase as in typical serine proteases. Taken together, the results obtained by the present study demonstrate that VSPase is a typical glutamate-specific serine endopeptidase.