Innate immunity is critical to insects and plays an important role in pathogen elimination and wound healing. Toll signaling pathway is the major signaling pathway associated with insect innate immunity mediating synthesis of anti-fungal/bacterial peptides. To better understand the Toll signaling pathway mediated immune response in Bombyx mori against Beauveria bassiana infection, expression patterns of genes encoding sixteen putative components of Toll signaling pathway in the silkworm larvae challenged with B. bassiana, including four pattern recognition receptors (PRRs, i.e. BmβGRP 1, 2, 3, 4), eight Toll-like receptors (TLRs, i.e. Bm18w, BmToll 1, 3, 6, 9, 7, 10, 11) and four effectors (BmMoricin 1, BmGloverin 2, BmDefensin 1 and BmLysozyme 1), were analyzed using quantitative real-time PCR. At the same time, the changes in their expression by RNAi knock-down of the four PRRs were also detected. Moreover, the effects of Toll signaling pathway inhibitors on antifungal activity in larvae hemolymph were also analyzed. The results showed that the expression levels of genes encoding sixteen putative components of Toll signaling pathway were obviously altered by the challenge with B. bassiana, but their temporal regulation mode was significantly different. Based on the expression patterns of the genes related to Toll signaling pathway, two sub-paths of immune signal recognition and transduction might be proposed in the response of silkworm larvae against B. bassiana infection. Besides, Toll signaling pathway inhibitor could significantly inhibit the antifungal activity in hemolymph and resulted in increased sensitivity of silkworm larvae to the B. bassiana infection, while the treatment with heat-inactivated B. bassiana could induce antifungal activity in the hemolymph and led to stronger resistance of the silkworm. These results implied that Toll signaling pathway played important roles in the antifungal immune response of the silkworm larvae, in which different components of Toll signaling pathway might play a specific regulatory function. These findings yield insights into the innate immune mechanisms underlying Toll signaling pathway in silkworm.