<jats:title>Abstract</jats:title><jats:p>As an attempt to search for bioactive natural products exerting anti‐inflammatory activity, we have evaluated the anti‐inflammatory effects of euscaphic acid (19α‐hydroxyursane‐type triterpenoids, EA) isolated from roots of <jats:italic>Rosa rugosa</jats:italic> and its underlying molecular mechanisms in lipopolysaccharide (LPS)‐induced RAW 264.7 macrophages. EA concentration‐dependently reduced the production of nitric oxide (NO), prostaglandin E<jats:sub>2</jats:sub> (PGE<jats:sub>2</jats:sub>), tumor necrosis factor‐α (TNF‐α), and interleukin‐1β (IL‐1β) induced by LPS in RAW 264.7 macgophages. Consistent with these data, expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) protein and iNOS, COX‐2, TNF‐α, and IL‐1β mRNA were inhibited by EA in a concentration‐dependent manner. In addition, EA attenuated LPS‐induced DNA binding and transcriptional activity of nuclear factor‐kappa B (NF‐κB), which was accompanied by a parallel reduction of degradation and phosphorylation of inhibitory kappa Bα (IκBα) and consequently by decreased nuclear translocation of p65 subunit of NF‐κB. Pretreatment with EA significantly inhibited the LPS‐induced phosphorylation of IκB kinase β (IKKβ), p38, and JNK, whereas the phosphorylation of ERK1/2 was unaffected. Furthermore, EA interfered with the LPS‐induced clustering of TNF receptor‐associated factor 6 (TRAF6) with interleukin receptor associated kinase 1 (IRAK1) and transforming growth factor‐β‐activated kinase 1 (TAK1). Taken together, these results suggest that EA inhibits LPS‐induced inflammatory responses by interference with the clustering of TRAF6 with IRAK1 and TAK1, resulting in blocking the activation of IKK and MAPKs signal transduction to downregulate NF‐κB activations. J. Cell. Biochem. 113: 1936–1946, 2012. © 2012 Wiley Periodicals, Inc.