Tussilagone (TSL), isolated from the flower of buds of Tussilago farfara (Compositae), is a sesquiterpenoid that is known to exert a variety of pharmacological activities. In the present study, we demonstrated that TSL exerts anti-inflammatory activities in murine macrophages by inducing heme oxygenase-1 (HO-1) expression. Treatment of RAW264.7 cells with TSL-induced HO-1 protein expression in a dose- and time-dependent manner without the induction of HO-1 mRNA expression. TSL-mediated HO-1 protein induction was not inhibited by treatment with actinomycin D, a transcriptional inhibitor, but by cycloheximide, a translational inhibitor. Moreover, mitogen-activated protein kinases (MAPKs) inhibitors such as SB203580, SP600125, and U0126 did not block TSL-mediated HO-1 protein expression, suggesting that the TSL-mediated HO induction may be regulated at the translational level. Consistent with the notion that HO-1 has anti-inflammatory properties, TSL inhibited the production of nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and prostaglandin E2 (PGE2) as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and murine peritoneal macrophages. Inhibition of HO-1 activity by treatment with zinc protoporphyrin IX (ZnPP), a specific HO-1 inhibitor, abrogated the inhibitory effects of TSL on the production of NO and PGE2 in LPS-stimulated RAW264.7 cells. Taken together, TSL may be an effective HO-1 inducer that has anti-inflammatory effects, and a valuable compound for modulating inflammatory conditions.