Abstract The Pregnane X receptor (PXR) is a member of the nuclear receptor family, which is highly expressed in liver as well as in intestine and regulates the transcription of genes involved in the metabolism, transport and elimination of xenobiotics. Recently, the role of PXR and xenobiotic metabolism in regulating inflammation has become evident. Here we demonstrate that in addition to its role in xenosensing and drug metabolism, PXR signaling is also essential for mediating innate antimicrobial immune responses. Mice lacking PXR were highly susceptible to the intracellular bacteria Listeria Monocytogenes (LM). Bone marrow derived macrophages (BMDMs) from PXR-/- mice were proficient in internalizing bacteria, but incapable of clearing LM. Surprisingly, the over activation of Toll-like receptor 4 (TLR4) signaling in the absence of PXR was the singular determinant of susceptibility in PXR-/- mice since mice that were deficient in both PXR and TLR4 were efficient in clearing LM. Furthermore, administration of TLR4 specific inhibitors rescued the ability of PXR-/- mice and BMDMs to clear bacteria. Our results demonstrate an unexpected role of PXR in controlling innate immunity by negatively regulating TLR4 gene expression. In addition, our study has uncovered a remarkable impact of TLR4 signaling in driving the quality of antimicrobial immune response to gram-positive bacteria.