Toll-like receptor 2 (TLR2) signaling has been shown to contribute to resistance to Listeria monocytogenes infection, as TLR2-deficient mice have a heightened susceptibility to infection with this organism. Because CD14 may associate with TLR2, we investigated the role of CD14 in Listeria responses. In both CD14-deficient and TLR2-deficient macrophages, nuclear factor kappaB translocation; CD40 and CD86; and the production of interleukin (IL)-12, IL-6, tumor necrosis factor, and nitric oxide are reduced. The absence of CD14 augmented susceptibility to Listeria infection, reduced survival, and diminished bacterial clearance, as observed in TLR2-deficient mice. Compared with C57BL/6 control mice, CD14-deficient mice were observed to have a greater number of hepatic microabscesses containing abundant neutrophils, these abscesses were larger in size, and there was reduced inducible nitric oxide synthase expression. Further, mice that are both CD14 deficient and TLR2 deficient display susceptibility to infection that is comparable to that of mice deficient in either CD14 or TLR2 alone. Therefore, the present data demonstrate the role of CD14 and TLR2 in the recognition and control of Listeria infection and host resistance.