Dioscoreanone, a 1,4-phenanthraquinone isolated from an ethanolic extract of the rhizome of Dioscorea membranacea, Pierre ex Prain & Burkill, a plant which has been used to treat inflammation and cancer in Thai Traditional Medicine. In this study, the mechanisms of dioscoreanone on LPS-induced NO production and cytokine expression through the activation of NF-κB and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess reagent, dioscoreanone was found to reduce NO levels with an IC50 value of 2.50 ± 0.64 μM, due to the significant suppression of LPS-induced iNOS mRNA expression, as well as IL-1β and IL-6 levels at a concentration of 6 μM. At the signal transduction level, using the pNFκB-Luciferase reporter system, dioscoreanone significantly inhibited NF-κB transcriptional activity, which resulted from the prevention of IκBα degradation. In addition, dioscoreanone in the range of 1.2-5 μM significantly enhanced LPS-induced ERK1/2 activation and dioscoreanone alone induced the activation of ERK1/2 proteins in a concentration- and time-dependent response. The activation of ERK1/2 proteins by dioscoreanone was due to both an arylating reaction, which was suppressed by N-acetyl cysteine, and a redox cycling reaction of NQOR, which was inhibited by dicoumarol. In conclusion, the mechanisms of dioscoreanone on the inhibition of NO production and mRNA expression of iNOS, IL-1β, and IL-6 were due to both the inhibition of NF-κB activation and the activation of ERK1/2 proteins. The activation of dioscoreanone may in turn inhibit the binding of NF-κB to pro-inflammatory gene promoters in LPS-induced RAW264.7 macrophage cells. J. Cell. Biochem. 113: 3427-3435, 2012. © 2012 Wiley Periodicals, Inc