Abstract Repeated exposure to low doses of endotoxin results in progressive hyporesponsiveness to subsequent endotoxin challenge, a phenomenon known as endotoxin tolerance. In spite of its clinical significance in sepsis and characterization of the TLR4 signaling pathway as the principal endotoxin detection mechanism, the molecular determinants that induce tolerance remain obscure. We investigated the role of the TRIF/IFN-β pathway in TLR4-induced endotoxin tolerance. Lipid A-induced homotolerance was characterized by the down-regulation of MyD88-dependent proinflammatory cytokines TNF-α and CCL3, but up-regulation of TRIF-dependent cytokine IFN-β. This correlated with a molecular phenotype of defective NF-κB activation but a functional TRIF-dependent STAT1 signaling. Tolerance-induced suppression of TNF-α and CCL3 expression was significantly relieved by TRIF and IFN regulatory factor 3 deficiency, suggesting the involvement of the TRIF pathway in tolerance. Alternatively, selective activation of TRIF by poly(I:C)-induced tolerance to lipid A. Furthermore, pretreatment with rIFN-β also induced tolerance, whereas addition of IFN-β-neutralizing Ab during the tolerization partially alleviated tolerance to lipid A but not TLR2-induced endotoxin homo- or heterotolerance. Furthermore, IFNAR1−/− murine embryonal fibroblast and bone-marrow derived macrophages failed to induce tolerance. Together, these observations constitute evidence for a role of the TRIF/IFN-β pathway in the regulation of lipid A/TLR4-mediated endotoxin homotolerance.