Objective To explore the interaction between 6 kD early secretory antigenic target (ESAT6) and autophagy in order to provide an experimental basis for the immune evasion mediated by ESAT6. Methods RAW264.7 cell lines (mouse macrophages) were treated with Earle's balanced salt solution (EBSS), and another cells were transfected by control plasmid pCMV-HA or recombinant plasmid pCMV-HA-ESAT6. Then we observed the growth of Bacillus Calmette-Guerin (BCG) in macrophages. Western blotting was used to detect the LC3 levels in RAW264.7 cells at 0, 8, 12, 32 hours after transducted by pCMV-HA-ESAT6. In RAW264.7 cells transfected with PCMV-HA, PCMV-HA-ESAT6, and treated with chloroquine (CQ) and CQ combined with pCMV-HA-ESAT6, which were lysed and cultured in Lowenstein-Jensen culture medium for BCG counting, LC3 was detected by Western blot analysis, and the number and size of lysosomes were observed by LysoTracker Red staining. Results Compared with control plasmid pCMV-HA transfected RAW264.7 cells, the number of BCG significantly increased in PCMV-HA-ESAT6-transfected cells, while decreased in EBSS-treated cells. PCMV-HA-ESAT6 transfection resulted in the increased transition of LC3 I to LC3 II in a time-depended manner. Compared with the controls, LysoTracker Red staining showed PCMV-HA-ESAT6, CQ and CQ plus PCMV-HA-ESAT6 transfections resulted in the increased number and size of lysosomes, and there were no differences among the three groups. Moreover, the growth potential of BCG was strong in the three transfection groups. Conclusion ESAT6 can inhibit the autophagy and promote the growth of BCG in RAW264.7 cells.