Abstract: Sargassum hemiphyllum, a kind of brown seaweed generally found along coastlines in East Asia, has long served as a traditional Chinese medicine. S. hemiphyllum has shown an anti-inflammatory effect; however, its mechanism has not been elucidated clearly. This study explored S. hemiphyllum for its biomedical effects. S. hemiphyllum sulfated polysaccharide extract (SHSP) was first prepared; the mouse macrophage cell line (RAW 264.7) activated by lipopolysaccharide (LPS) was used as a model system. The secretion profiles of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, and NO, were found significantly to be reduced in 1−5 mg/mL dose ranges of SHSP treatments. RT-PCR analysis suggested SHSP inhibits the LPS-induced mRNA expressions of IL-β, iNOS, and COX-2 in a dose-dependent manner. At protein levels, Western blot analysis demonstrated a similar result for NF-κB (p65) in cytosol/nuclear. Taken together, the anti-inflammatory properties of SHSP may be attributed to the down-regulation of NF-κB in nucleus.