The anti-inflammatory mechanism of 5-hydroxy-3,6,7,8,3′,4′- hexamethoxyflavone (5HHMF), a polyhydroxyflavone isolated from the marine algae Hizikia fusiforme, was investigated in RAW 264.7 murine macrophage cells. Western blot and reverse transcriptase PCR analyses indicated that adding 5HHMF to cultured cells significantly reduced the production of nitric oxide and prostaglandin E2 and downregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In addition, 5HHMF inhibited the release of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β, and decreased the transcriptional levels. In particular, 5HHMF significantly inhibited the LPS-induced nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus, which was associated with the abrogation of inhibitory IκBα degradation and subsequent decreases in nuclear p65 levels. In conclusion, these results suggested that the anti-inflammatory activities of 5HHMF may be attributed to the inhibition of iNOS, COX-2 and cytokine expression by attenuating NF-κB activation via IκBβ degradation in macrophages.