Technical Abstract: Toll-like receptors can activate two distinct branches of downstream signaling pathways: MyD88 and TRIF pathways lead to the expression of pro-imflammatory cytokines and type I interferon genes, respectively. Numerous reports have demonstrated that resveratrol, a phytoalexin with anti-inflammatory effects, inhibits NFkB activation and other downstream signaling pathways leading to the suppression of target gene expression. However, the direct targets of resveratrol have not been identified. Here, we attempted to identify the molecular target for resveratrol in TLR-mediated signaling pathways. Resveratrol suppressed NFkB activation and cyclooxygenase-2 expression in RAW264.7 cells following TLR3 and TLR4 stimulation, but not TLR2 or TLR9. Further, resveratrol inhibited NFkB activation induced by TRIF, but not by MyD88. The activation of IRF3 and the expression of IFNb induced by LPS, polyI:C, or TRIF were also suppressed by resveratrol. The suppressive effect of resveratrol on LPS-induced NFkb activation was abolished in TRIF-deficient mouse embryonic fibroblasts while LPS-induced degradation of IkBa and expression of cyclooxygenase-2 and iNOS were still inhibited in MyD88-deficient macrophages. Futhermore, resveratrol inhibited the kinase activity of TBK1 and the NFkB activation induced by RIP1 in RAW264.7 cells. Together, these results demonstrate that resveratrol specifically inhibits TRIF signaling in the TLR3 and TLR4 pathway by targeting TBK1 and RIP1 in TRIF complex. The results raise the possibility that certain dietary phytochemicals can modulate TLR-derived signaling and inflammatory target gene expression, and alter susceptibility to microbial infection and chronic inflammatory diseases.