TRAIL receptor (TRAIL-R) signaling has been implicated in inducing apoptosis in tumor cells, but little is understood about its physiological function. Here, we report the generation and characterization of TRAIL-R^−/− mice, which develop normal lymphocyte populations but possess enhanced innate immune responses. TRAIL-R^−/− mice exhibited increased clearance of murine cytomegalovirus that correlated with increased levels of IL-12, IFN-α, and IFN-γ. Stimulation of macrophages with Mycobacterium and Toll-like receptor (TLR)-2, -3, and -4, but not TLR9, ligands resulted in high levels of TRAIL upregulation and enhanced cytokine production in TRAIL-R^−/− cells. The immediate-early TLR signaling events in TRAIL-R^−/− macrophages and dendritic cells are normal, but IκB-α homeostatic regulation and NF-κB activity at later time points is perturbed. These data suggest that TRAIL-R negatively regulates innate immune responses.