The genus of staphylococcus widely distributes in environments and contributes to a variety of animal and human diseases. The enterotoxins (SEs) secreted by this type of pathogen have been the leading cause of bacterial toxic shock syndrome and food poisoning, and thus present a substantial concern to public health. In this study, we analyzed the superantigen profile of 122 staphylococcus strains isolated from diverse sources. When screened for the presence and prevalence of 17 known se or se-like (sel) genes, except selj, all other genes were detected in these isolates. In particular, 95.9% of the isolates harbored at least one se/sel gene. Moreover, 47.5% of them bore at least 5. Remarkably, several non-pathogenic species of animal- and environment-origin were also found to carry multiple se/sels. The most frequent genes detected were tsst (62.3%), sei (54.1%), and seb (46.7%), followed by some sel genes (selo, selu, and selm), which also were present at relatively high frequency (20-30%). The generated data improved understanding of strain-specific differences in enterotoxin expression. The gene products of the latter (selo and selu) were subsequently analyzed for their antigenicity in a mouse model using purified E. coli-based recombinant proteins. The studies revealed a strong activity for SEO in induction of T-lymphocyte proliferation and production of various inflammatory cytokines either in vivo or in vitro. In contrast, SEU exhibited little superantigenic effects. The molecular basis for the difference in antigenicity was analyzed by 3D homology remodeling, which revealed a difference in binding and affinities for MHC-II molecules and TCR Vβ region.