We have investigated the role of Toxoplasma gondii–derived heat shock protein 70 (TgHSP70) as a B cell mitogen by measuring proliferative responses in vitro. TgHSP70 induced prominent proliferative responses in murine B cells derived not only from T gondii–infected but also from uninfected mice. Nude mice responded to TgHSP70; however, severe combined immunodeficiency, RAG1^−/− B6, and μMT mice failed to respond. B220 spleen cells showed marked proliferation after stimulation with TgHSP70, but neither CD4 nor CD8 population responded. This unresponsiveness of CD4 and CD8 T cells to TgHSP70 was antigen presenting cells independent. These data indicate that TgHSP70 induced the proliferation of B cells but not T cells. Polymyxin B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate TgHSP70-induced proliferation. C3H/HeN mice responded well to TgHSP70 stimulation; however, C3H/HeJ mice carrying a point mutation in the Toll-like receptor (TLR) 4 failed to respond. This indicates that TLR4 is required for TgHSP70-induced B cell activation. The involvement of TLR4 in the TgHSP70-induced proliferative responses of spleen cells was also shown by the use of TLR4^−/− mice. But TgHSP70-induced, but not LPS-induced, spleen cell proliferation was observed in MyD88^−/− mice, indicating that the MyD88 molecule was involved in LPS-induced proliferation but not in TgHSP70-induced proliferation.