Capsaicin and dihydrocapsaicin were extracted from dry chili with the solvent of n–hexane as and separated by high–speed countercurrent chromatography. Chemical structures of capsaicin and dihydrocapsaicin were determined by LC–MS and NMR. Anti–inflammatory activity of the two compounds were evaluated by examining the effect on NO release of RAW264.7 macrophages. The result showed that the best solvent system was n–hexane: ethyl acetate∶methanol∶water∶ice acetic acid = 15∶15∶20∶20∶7.5 (V/V). The upper phase was used as the stationary phase and the lower phase was used as the mobile phase, which was performed at a flow rate of 2 mL /min. The apparatus rotated at 850 r/min, detected at 270 nm and the separation temperature was 25 ℃. Within 300 min, the crude extract was injected for twice with 300 mg at each time. 22 mg compound 1 with a purity of 98.2% and 16 mg compound 2 with a purity of 98.0% were obtained. The mass spectrometry and nuclear magnetic resonance (NMR) indicated that compound 1 was dihydro capsaicin and that compound 2 was capsaicin. The IC50 inhibitory effects of dihydrocapsaicin and capsaicin on the NO release of RAW264.7 macrophages were（42.66 ±4.88）and (25.68±3.86) μmol/L, respectively, both were lower than that of the positive control indomethacin (127.88±2.45) μmol/L. Therefore, the anti–inflammatory effect of capsaicin was the best, dihydrocapsaicin second and indomethacin the worst.