Sargassum fulvellum (Turner) C. Agardh has been used to treat various inflammatory diseases, including lump, dropsy, swollen and painful scrotum, and urination problems for several centuries with no side effects. This study aims to investigate the anti-inflammatory effect of the hexane fraction of S. fulvellum (HFS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and phorbol 12-myristate 13-acetate (PMA)-induced mouse-ear edema. The anti-inflammatory activity of HFS in LPS-stimulated RAW 264.7 cells was investigated by assessing the inhibition of nitric oxide (NO) and pro-inflammatory cytokine production during Griess reaction and enzyme-linked immunosorbent assay (ELISA), respectively. The molecular mechanisms that underlie the anti-inflammatory action of HFS were investigated by analyzing the activation of transcription factor and its upstream signaling proteins. Additionally, an in vivo study of the anti-inflammatory effect of HFS was carried out using PMA-induced mouse-ear edema. HFS inhibited LPS-induced NO production in a dose-dependent manner and suppressed the expression of inducible NO synthase (iNOS) in the RAW 264.7 cells. Further, HFS reduced the production of pro-inflammatory cytokines in the LPS-stimulated RAW 264.7 cells. HFS significantly inhibited LPS-induced nuclear factor kappa B (NF-κB) transcriptional activity and NF-κB translocation into the nucleus by preventing degradation of inhibitor κB-α. Moreover, HFS inhibited the activation of Akt and mitogen-activated protein kinases (MAPKs) in the LPS-stimulated RAW 264.7 cells. Furthermore, HFS suppressed PMA-induced mouse-ear edema. The above data indicate that the anti-inflammatory effects of HFS on LPS-stimulated cells are associated with the suppression of NF-κB through the inhibition of MAPKs and Akt phosphorylation.