Sortase mediated transpeptidation reactions play a significant role in covalent attachment of surface proteins to the cell wall of Gram-positive bacteria. Earlier studies have shown that sortase A (StrA) is required for the virulence of Staphylococci. The human pathogen Staphylococcus simulans CJ16 carries a putative sortase A (SsiStrA) encoding gene， but neither transpeptidation activity nor biochemical characteristics of SsiStrA have been investigated. Here， we identified and characterized StrA from coagulase-negative Staphylococci. SsiStrA was cloned and overexpressed in Escherichia coli BL21 in a soluble form. Size-exclusion chromatography， cross-linking and dynamic light scattering demonstrated that SsiStrA existed as monomer-dimer equilibrium in vitro. We further demonstrated that SsiStrA has sortase activity， and it recognized and cleaved the sorting motif LXPTG. H117， C180 and R193 residues were critical for enzyme activity， and calcium ions enhanced activity.