Myagropsis myagroides has been used as a Chinese medicine and its extract has shown various biological activities, however, its anti-inflammatory mechanism remains unknown. In this study, we investigated the inhibitory effects of the ethyl acetate fraction of M. myagroides (EFM) on the production of inflammatory mediators and pro-inflammatory cytokines in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells. EFM significantly inhibited LPS-induced production of nitric oxide (NO), prostaglandin E(2), and pro-inflammatory cytokines in a dose-dependent manner and suppressed the production of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 in RAW 264.7 cells. Inhibitory effect of EFM on iNOS expression and NO production was further confirmed using LPS-activated mouse peritoneal macrophages. EFM treatment strongly suppressed the activation of nuclear factor-kappa B (NF-κB) by suppressing phosphorylation of Akt and extracellular signal-regulated kinases (ERKs). EFM as well as phlorofucofuroeckol B (PFF-B), a major compound isolated from EFM, reduced ear edema induced by phorbol 12-myristate 13-acetate in mice. These results indicate that the anti-inflammatory effect of EFM, rich in PFF-B, on LPS-stimulated macrophages is regulated by the inhibition of NF-κB pathway through the inhibition of ERKs and Akt phosphorylation in LPS-stimulated macrophage cells.