Technical Abstract: Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NF'B and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of chronic inflammatory and immune diseases. Cinnamaldehyde (3-phenyl-2-propenal), has been reported to inhibit NF'B activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NF'B and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNB in macrophages (RAW264.7). Cinnamaldehyde inhibited NF'B activation induced by constitutively active TLR4 or wild-type TLR4 while it showed no effect on the activation of NFkB or IRF3 induced by MyD88, IKKB, TRIF or TBK1. Ligand-dependent oligomerization of TLR4 was suppressed by cinnamaldehyde resulting in the downregulation of NFkB activation. Our results demonstrated that the molecular target of cinnamaldehyde is the oligomerization process of TLR4, but not MyD88-dependent nor TRIF-dependent downstream components, leading to the suppression of the activation of TLR-signaling pathways and the consequent expression of inflammatory target genes.