4'-Hydroxywogonin (4'-HW), a flavonoid, has been isolated from various plants and shown to inhibit NO production in macrophages. However, the molecular mechanisms and its in vivo activity have not been determined. Our study aimed to investigate the mechanisms underlying the anti-inflammatory effects of 4′-HW in vitro and in vivo. We showed that 4′-HW potently reduced the expression levels of COX-2 and iNOS as well as their products, prostaglandin E (PGE) and nitric oxide (NO) respectively, in LPS-stimulated RAW 264.7 macrophages. 4′-HW also suppressed LPS-induced pro-inflammatory cytokines at mRNA and protein levels. Moreover, 4′-HW blocked the interaction of TAK1 and TAB1 in LPS-stimulated RAW 264.7 macrophages, resulting in an inhibition of the TAK1/IKK/NF-κB signaling pathway. Furthermore, 4′-HW also reduced the phosphorylation of MAPKs and PI3/Akt signaling pathways in LPS-stimulated RAW 264.7 macrophages. 4′-HW was also significantly decreased the intracellular reactive oxygen species (ROS) level. The effect of 4′-HW was confirmed in vivo. 4′-HW exhibited potent protective effect against LPS-induced ALI in mice. These findings indicate that 4′-HW suppresses the LPS-induced response in vitro and in vivo. It is likely that the inhibition of the TAK1/IKK/NF-κB, MAPKs and PI3/AKT signaling pathways contribute to the anti-inflammatory effects of 4′-HW. Our study suggests that 4′-HW may be an important functional constituent in the plants and has the potential value to be developed as a novel anti-inflammatory agent.