The anti-inflammatory activity of fruit body of Phellinus pini was investigated by activity-guided fractionation. From the screening of each fraction for the inhibitory activity of NO production in lipopolysaccaride (LPS) activated RAW 264.7 cells, methanol extract and its hexane soluble fraction of Phellinus pini exhibited inhibition of NO production compared with LPS control without toxicity. The hexane soluble fraction showed dose-dependent inhibition of NO production. The active hexane fraction was repeatedly chromatographed over silica gel, ergosta-7,24(28)-dien-3-ol(1) and ergosterol peroxide (2) were isolated and identified. Ergosterol derivatives were inhibited NOS activation, IC50 of them were 18.9±3.9 μM (1) and 20.4±4.5 uM (2).