Chalcones have anti-inflammatory properties. Here, we synthesized 2'-methoxy-4'6'-bis(methoxymethoxy)chalcone (MBMC) and examined its anti-inflammatory effects. MBMC inhibited nitric oxide production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. MBMC also blocked LPS-induced activation of nuclear factor kappaB (NF-kappaB), p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK). MBMC increased haem oxygenase 1 (HO-1) expression and nuclear accumulation of nuclear factor-erythroid 2-related factor 2 (Nrf2), an essential transcription factor for HO-1 induction. Treatment with tin protoporphyrin, a selective inhibitor of HO-1, reversed the inhibition of nitric oxide production by MBMC, suggesting that HO-1 induction mediates MBMC-mediated suppression of nitric oxide production. MBMC treatment rapidly and transiently decreased glutathione (GSH) levels, and treatment with GSH-Et (cell permeable form of GSH) or N-acetylcysteine (precursor of GSH) counteracted the HO-1 and Nrf2 expression elicited by MBMC, indicating that MBMC-induced HO-1 expression requires transient depletion of GSH. In summary, MBMC inhibits LPS-stimulated nitric oxide production via down-regulation of inflammatory pathways (NF-kappaB, p38 and JNK) and induction of the protective enzyme, HO-1.