Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host. In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection. Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-γ production, leading to normal LM clearance in the host. IFN-γ knockout (KO) mice were highly susceptible to LM infection. IL-12/IL-18 double knockout mice were also highly susceptible. Their susceptibility was less than that of IFN-γ KO mice, but more than that of single IL-12 or IL-18 KO mice. Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-γ following LM infection and were most susceptible to LM. Furthermore, MyD88-deficient, but not IFN-γ-deficient, Kupffer cells could not produce TNF-α in response to LM in vitro, indicating the importance of MyD88-dependent TNF-α production for host defense. As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-γ, and TNF-α, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-γ production. These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-α production and for IL-12-and IL-18-mediated IFN-γ production in early phase clearance of LM.