Staphylococcal enterotoxin C2 (SEC2) is one of the most potent known activators of human T lymphocytes, and recombinant SEC2 shows promising clinical values, but SEC2 can cause food poisoning and toxic shock syndrome in vivo. In this study, site-directed mutagenesis has been used to introduce alanine substitutions at Phe144 and Leu45 in the molecule. The mutant genes were cloned and expressed, and the corresponding proteins were purified by nickel agarose affinity chromatography. We found that the SEC2 mutant proteins could stimulate the proliferation of human peripheral blood lymphocytes and inhibit the growth of tumour cells as native SEC2. Furthermore, flow cytometry assay showed that mSEC2(F44A, L45A) drastically reduced the ability of the toxin to bind to MHC class II. Physiological parameters revealed that mSEC2(F44A, L45A) reduced significantly rat temperature compared with native SEC2 in vivo. Our results clearly suggest that this genetically modified SEC2 protein is less toxic and justifies its further development as a new, safer antitumour superantigen to prevent SEC2 intoxication.