Objective To investigate the anti-inflammatory activity of dehydrocostus lactone (DCL) from natural plant wood fragrance on lipopolysaccharide (LPS)/interferon-gamma (IFNγ)-stimulated RAW264.7 macrophages and to determine the molecular mechanism of the anti-inflammatory activity of DCL. Methods RAW264.7 macrophages were stimulated with LPS (0.5 μg/ml) and IFNγ (10 ng/ml), and then treated with different concentrations of DCL. Treated RAW264.7 cells were then detected the nitric oxide (NO) production, the prostaglandin E2 (PGE2) and their synthesized upstream enzyme Inducible Nitric Oxide Synthase (iNOS) and Cyclooxygenase-2 (COX-2). Finally, we also detected key target proteins of Nuclear Factor Kappa-B (NF-κB) signaling pathway by western blot. Results Without exhibiting significant cytotoxicity, DCL inhibited the production of NO and PGE2 induced by LPS/IFNγ in RAW264.7 macrophages in a dose-dependent manner. Additionally, DCL suppressed the protein expression levels of iNOS and COX-2 in LPS/IFNγ-stimulated RAW264.7 macrophages, demonstrating notable anti-inflammatory activity of DCL. Furthermore, DCL significantly inhibited the phosphorylation of classic NF-κB signaling pathway key proteins, including inhibitor of Nuclear Factor Kappa-B Kinaseα/β (IKKα/β) and inhibitor of Nuclear Factor Kappa-Bα (IκBα), activated during the inflammatory response of RAW264.7 macrophages. Conclusion DCL exerts anti-inflammatory activity on macrophages by inhibiting the classical NF-κB pathway, providing a new therapeutic strategy for the treatment of inflammatory diseases related to NF-κB pathway.