Background: Immunotherapy has been used to improve patient immune function, inhibit tumor growth and has become a highly promising method of cancer treatment.Highly agglutinative staphylococcin (HAS), a mixture of Staphylococcus aureus culture filtrates, which include staphylococcal enterotoxin (SE) C as the active ingredient, has been used clinically as an immunomodifier in the treatment of a number of tumors for many years.However, the use of HAS has been associated with some unavoidable side-effects such as fever.Previous studies have shown that SEB stimulates a more potent activation of T lymphocytes than SEC3, and mutations of the histidine residues eliminated the toxicity of SEB.SE mutants with decreased side-effects and/or more potent antitumor activities are required.Methodology/Principal Findings: We built a structural model of the MHC Ⅱ-SEB-TCR complex and found that a mutation of SEB at Lys173 might decrease the repulsion force between the SEB-TCR, which would facilitate their interaction.From the above results, we designed SEB-H32Q/K173E (mSEB).Analysis of in vitro stimulation of the proliferation of human peripheral blood mononuclear cells (PBMCs), IFN-y secretion and inhibition of the growth of various tumor cell lines demonstrated that mSEB exhibited higher antitumor activity compared with wild-type SEB (wtSEB).Notably, mSEB inhibited the growth of various tumors at an extremely low concentration with little cytotoxicity against normal cells.Three animal tumor models (C57BL/6 mouse, New Zealand rabbit and a humanized NOD/SOD mouse) were used to evaluate the in vivo immunotherapeutic effects.Compared with WtSEB, mSEB significantly enhanced antitumor effect in more than one animal model with reduced pyrexia toxicity and prolonged the survival of tumor-bearing mice.Conclusíons/Sígnificance: Our results suggest that SEB-H32Q/K173E retains superantigen (Sag) characteristics and enhances the host immune response to neoplastic diseases while reducing associated pyrogenic toxicity.