Objective To explore the effect of MPT64 antigen from Mycobacterium tuberculosis on RAW264.7 macrophages and the related mechanism. Methods MPT64 was purified after expression in E.coli and verified by Western blotting analysis. The RAW264.7 differentiation was induced by phorbol myristate acetate (PMA) and the resultant cells were divided into three groups according to different treatments: negative control, purified protein derivative of BCG(BCG-PPD) and BCG-PPD+MPT64 treatment groups. After 16 h incubation, flow cytometry was used to examine apoptosis of macrophages, and the levels of TNF-α and IL-10 in the supernatants were determined by ELISA. Results BCG-PPD treatment induced apoptosis of RAW264.7 macrophages, and compared with BCG-PPD group, the apoptotic level of macrophages was significantly lower in BCG-PPD+MPT64 group (P<0.05). We also found that the supernatant TNF-α level in the BCG-PPD group was significantly higher than that in negative group (P<0.01) and the IL-10 levels were not significantly defferent between the two groups. Compared with BCG-PPD group, the IL-10 level was significantly increased in BCG-PPD+MPT64 group (P<0.01) and the TNF-α levels were not significantly different between the two groups. Conclusion MPT64 may act as a virulence factor and can inhibit the apoptosis of macrophages induced by BCG-PPD, which is probably through increasing IL-10 level.