The medical use of bee venom for rheumatoid arthritis (RA) has a very long tradition. In this study, isolation and purification of polypeptides from bee venom were carried out on sephadex chromatography, heparin sepharose CL-6B chromatography and HPLC. Several fractions were extracted, and their effects on activation of splenocyte and THP-1 cell were studied. The inhibitory fraction was selected for further studies. Finally, BV I -2H that the HPLC elution profiles was a single peak was isolated by C8 column. ESIMS detection results showed that BV I -2H was a fraction of bee venom, and the molecular weight of the major component was 644.8. BV I -2H could inhibit ConA-induced splenocyte proliferation, IL-1 production and interfere with splenocyte cycle in mice. Moreover, BV I -2H could inhibit PMA-induced TNFα production in THP-1 cells, which was due to its inhibitory effects on TNFα mRNA expression and protein phosphorylation of IκBα. Our studies indicated that BV I -2H was one of the anti-inflammatory components of bee venom.