Vα14 natural killer T (iNKT) cells activated by alpha-galactosylceramide (GalCer) secrete a large amount of cytokines. Toll-like receptors (TLRs) play a critical role in the innate immune responses via the recognition of pathological antigen. Previously we demonstrated that the iNKT cells activated by GalCer augmented LPS-induced NO production in peritoneal cells. In this study, we examined the effect of GalCer and TLR agonists by IFN-γ production from splenocytes. Splenocytes pretreated with GalCer induced TLR3, 4, 7/8, and 9 agonists in vitro, resulting in the enhancement of IFN-γ mRNA expression. In particular, IFN-γ stimulated by GalCer and LPS was increased in NK cells and CD8 T cells, and inhibited by a neutralizing anti-IL-12 antibody. Pretreatment with GalCer enhanced the phosphorylation of IκB-α induced by LPS stimulation. The present study showed that co-stimulation of GalCer and TLR agonists powerfully induced the production of IFN-γ from splenocytes.