Toll-like receptors (TLRs) play important roles in induction of innate immune responses for both host defense against invading pathogens and wound healing after tissue injury. Since dysregulation of TLR-mediated immune responses is closely linked to many chronic diseases, modulation of TLR activation by small molecules may have therapeutic potential against such diseases. Expression of the majority of lipopolysaccharide-induced TLR4 target genes is mediated through a MyD88-independent (TRIF-dependent) signaling pathway. In order to evaluate the therapeutic potential of the flavonoid luteolin we examined its effect on TLR-stimulated signal transduction via the TRIF-dependent pathway. Luteolin suppressed activation of Interferon regulatory factor 3 (IRF3) and NFkappaB induced by TLR3 and TLR4 agonists resulting in the decreased expression of target genes such as TNF-alpha, IL-6, IL-12, IP-10, IFNbeta, CXCL9, and IL-27 in macrophages. Luteolin attenuated ligand-independent activation of IRF3 or NFkappaB induced by TLR4, TRIF, or TBK1, while it did not inhibit TLR oligomerization. Luteolin inhibited TBK1-kinase activity and IRF3 dimerization and phosphorylation, leading to the reduction of TBK1-dependent gene expression. Structural analogs of luteolin such as quercetin, chrysin, and eriodictyol also inhibited TBK1-kinase activity and TBK1-target gene expression. These results demonstrate that TBK1 is a novel target of anti-inflammatory flavonoids resulting in the down-regulation of the TRIF-dependent signaling pathway. These results suggest that the beneficial activities of these flavonoids against inflammatory diseases may be attributed to the modulation of TLR-mediated inflammatory responses.