Abstract: Objective  To evaluate the immunogenicity of the recombinant M. smegmatis expressing Ag85B-ESAT-6 fusion protein of M. tuberculosis (Ag85B-ESAT-6-rM.s) in mice.  Methods  Six-week-old BALB/c mice were immunized by subcutaneous injection on the back with 1×10⁶ colony-forming unit (CFU) of Ag85B-ESAT-6-rM.s per mouse. The mice immunized by M. smegmatis (M.s), BCG, Ag85B-ESAT-6 fusion protein and saline used as control. The percentages of CD4⁺ and CD8⁺ T cells in the peripheral blood mononuclear cells (PBMC) of mice were detected once a week for six weeks after immunization. The proliferation of splenic lymphocytes of mice was assayed by MTS［3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt］, and the numbers of splenic lymphocytes secreting interferon γ (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4) were detected by enzyme-linked immunospot assay (ELISPOT) at 6 weeks after immunization.  Results  The Ag85B-ESAT-6-rM.s could significantly stimulate the generation of CD4⁺ and CD8⁺ T cells in the PBMC from immunized mice, and its percentages of CD4⁺ and CD8⁺ T cells ［(59.6±1.5)%］were significantly higher than that in BCG group ［(48.8±2.3)%］ (t=12.252,  P<0.05) and M.s group ［(45.7±1.6)%］(t=20.166,  P<0.05). Ag85B-ESAT-6-rM.s could stimulate the proliferation of splenic lymphocytes in immunized mice,  and its stimulation index (3.23±0.31) was equivalent to that in BCG group (2.95±0.36). However, the level of IFN-γ in mice immunized by Ag85B-ESAT-6-rM.s ［(167.5±36.6) spot forming cells (SFC)/10⁶］was significantly higher than that in BCG group ［(98.5±26.9)SFC/10⁶ ］(t=4.804,  P<0.05), and the level of IFN-γ was also higher than levels of IL-2 and IL-4 (t=6.174 and 6.449,  P<0.05) in mice immunized by Ag85B-ESAT-6-rM.s.  Conclusion  The expression of Ag85B-ESAT-6 fusion protein in M. smegmatis could improve the immunogenicity, and Ag85B-ESAT-6-rM.s could induce immune response preventing M. tuberculosis infection in mice. Ag85B-ESAT-6-rM.s as a TB candidate vaccine is worthy of further study.