The currently used vaccine against tuberculosis, Bacille Calmette-Guérin (BCG), has variable efficacy, so new vaccine development is crucial. In this study, we evaluated a recombinant vaccine prepared from non-pathogenic Mycobacterium smegmatis (rMS) that expresses a fusion of early secreted antigenic target 6-kDa antigen (ESAT6) and culture filtrate protein 10 (CFP10). C57BL/6 mice were immunized with the rMS expressing the ESAT6-CFP10 fusion protein (rM.S-e6c10) or with BCG. The mice in the rM.S-e6c10 group had a significantly higher titre of anti-ESAT6-CFP10 antibodies than did animals in the BCG or saline groups. Spleen cells from rM.S-e6c10-immunized mice exhibited a cytotoxic response to ESAT6 and CFP10-expressed target cells, but spleen cells from animals in the other groups did not. Levels of IFN-γ and IL-2 production by purified T cells from spleens were significantly higher in rM.S-e6c10 group than in BCG group. Finally, after M. tuberculosis (MTB)-challenged mice, dramatic reduction in the numbers of MTB colony-forming units (CFUs) in the lungs was observed for the mice immunized with the rMS. The protective efficacy of rM.S-e6c10 and BCG vaccination was similar based on measures of MTB burden and lung pathology. Our data indicate that the recombinant M. smegmatis vaccine expressing the ESAT6-CFP10 fusion protein has potential in clinic application.