Conserved bacterial components potently activate host immune cells through transmembrane Toll-like receptors (TLRs), which trigger a protective immune response but also may signal apoptosis. In this study, we investigated the roles of TLR2 and TLR4 as inducers of apoptosis in Yersinia enterocolitica-infected macrophages. Yersiniae suppress activation of the antiapoptotic NF-kappaB signaling pathway in host cells by inhibiting inhibitory kappaB kinase-beta. This leads to macrophage apoptosis under infection conditions. Experiments with mouse macrophages deficient for TLR2, TLR4, or both receptors showed that, although yersiniae could activate signaling through both TLR2 and TLR4, loss of TLR4 solely diminished Yersinia-induced apoptosis. This suggests implication of TLR4, but not of TLR2, as a proapoptotic signal transducer in Yersinia-conferred cell death. In the same manner, agonist-specific activation of TLR4 efficiently mediated macrophage apoptosis in the presence of the proteasome inhibitor MG-132, an effect that was less pronounced for activation through TLR2. Furthermore, the extended stimulation of overexpressed TLR4 elicited cellular death in epithelial cells. A dominant-negative mutant of Fas-associated death domain protein could suppress TLR4-mediated cell death, which indicates that TLR4 may signal apoptosis through a Fas-associated death domain protein-dependent pathway. Together, these data show that TLR4 could act as a potent inducer of apoptosis in macrophages that encounter a bacterial pathogen.