Numerous studies have examined the role of flavonoids in modulating inflammatory responses in vitro. In this study, we found a novel flavonoid, 3,6,3′-trihydroxyflavone (1), with anti-inflammatory effects. Antiinflammatory activity and mechanism of action were examined in mouse macrophages stimulated with lipopolysaccharide (LPS). Our results showed that the anti-inflammatory effects of 1 are mediated via p38 mitogen-activated protein kinase (p38 MAPK), Jun-N terminal kinase (JNK), and the extracellularsignal-regulated kinase (ERK) pathway in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Binding studies revealed that 1 had a high binding affinity to JNK1 (1.568 × 10 8 M −1) and that the 3-and 6-hydroxyl groups of the C-ring and A-ring of 1 participated in hydrogen bonding interactions with the side chains of Asn114 and Lys55, respectively. The oxygen at the 3′ position of the B-ring formed a hydrogen bond with side chain of Met111. Therefore, 1 could be a potential inhibitor of JNKs, with potent anti-inflammatory activity.