A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.