Hydroxylated polymethoxyflavones (PMFs) are a unique class of flavonoid compounds mainly found in citrus plants. We investigated the anti-inflammatory effects of one major 5-hydroxy PMF, namely 5-demethylnobiletin (5DN) and its metabolites 5, 3′-didemethylnobiletin (M1), 5, 4′-didemethylnobiletin (M2), and 5, 3′, 4′-tridemethylnobiletin (M3) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results showed that M2 and M3 produced stronger inhibitory effects on the production of nitric oxide (NO) than their parent compound at non-cytotoxic concentrations. Western blotting and real-time PCR analyses demonstrated that M2 and M3 significantly decreased iNOS and COX-2 gene expression. The results also showed that M1 and M3 induced heme oxygenase-1(HO-1) gene expression. Overall, our results demonstrated that metabolites of 5DN significantly inhibited LPS-induced inflammation in RAW 264.7 macrophage cells and generally possessed more potent anti-inflammatory activity than the parent compound, 5DN.