TAK-242 (resatorvid), a small-molecule-specific inhibitor of Toll-like receptor (TLR) 4 signaling, inhibits the production of lipopolysaccharide-induced inflammatory mediators by binding to the intracellular domain of TLR4. Cys747 in TLR4 has been identified previously as the binding site of TAK-242. However, the mechanism by which TAK-242 inhibits TLR4 signaling after binding to TLR4 remains unknown. The present study demonstrated, using coimmunoprecipitation, that TAK-242 interferes with protein-protein interactions between TLR4 and its adaptor molecules. Among 10 different human TLRs, TAK-242 selectively bound to TLR4. The time course of the inhibitory effect of TAK-242 on inflammatory mediator production corresponded to that of the binding of TAK-242 to TLR4. TAK-242 inhibited the association of TLR4 with Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) or Toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon-β-related adaptor molecule (TRAM) in human embryonic kidney (HEK) 293 cells overexpressing TLR4, MD-2, and TIRAP or TRAM, respectively. TAK-242 inhibited the TIRAP-mediated activation of nuclear factor κB (NF-κB) and the TRAM-mediated activation of NF-κB and interferon-sensitive response element in HEK293 cells stably expressing TLR4, MD-2, and CD14. The activation of endogenous interleukin-1 receptor-associated kinase in RAW264.7 cells was also inhibited by TAK-242 treatment. These findings suggest that TAK-242 binds selectively to TLR4 and subsequently disrupts the interaction of TLR4 with adaptor molecules, thereby inhibiting TLR4 signal transduction and its downstream signaling events. This work proposes a novel paradigm of a small molecule capable of disrupting protein-protein interactions.