Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/ Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01–100 mM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)–induced calcium influx, tumor necrosis factor-a, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter–like/myeloid differentiation primary response 88– and TRIF-related adaptor molecule/TIR domain–containing adapter-inducing interferon-b–dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain–containing adapter-inducing interferon-b knockout, and MyD88 adapter–like knockout macrophages with gedunin (10 mM) significantly inhibited LPS (50 ng/ml)– induced tumor necrosis factor-a and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 mM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor kB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.