Abstract: 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 κB (NF‐κB), 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‐κB, p38 and JNK) and induction of the protective enzyme, HO‐1.