Dioscorealide B (DB), a naphthofuranoxepin has been purified from an ethanolic extract of the rhizome of Dioscorea membranacea Pierre ex Prain & Burkill which has been used to treat inflammation and cancer in Thai Traditional Medicine. Previously, DB has been reported to have anti-inflammatory activities through reducing nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in lipopolysaccharides (LPS)-induced RAW 264.7 macrophage cells. In this study, the mechanisms of DB on LPS-induced NO production and cytokine expression through the activation of nuclear factor-κB (NF-κB) and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess's reagent, DB reduced NO level with an IC 50 value of 2.85±0.62 μM that was due to the significant suppression of LPS-induced iNOS mRNA expression as well as IL-1β, IL-6, and IL-10 mRNA at a concentration of 6 μM. At the signal transduction level, DB significantly inhibited NF-κB binding activity, as determined using pNFκB-Luciferase reporter system, which action resulted from the prevention of IκBα degradation. In addition, DB in the range of 1.5-6 μM significantly suppressed the activation of the ERK1/2 protein. In conclusion, the molecular mechanisms of DB on the inhibition of NO production and mRNA expression of iNOS, IL-1β, IL-6, and IL-10 were due to the inhibition of the upstream kinases activation, which further alleviated the NF-κB and MAPK/ERK signaling pathway in LPS-induced RAW264.7 macrophage cells. © 2010 Wiley-Liss, Inc.