The immunogenicity of the recombinant Bacille Calmette-Guerin: rBCG-Ag85B-Mpt64(190-198)-Mtb8.4 (rBCG-AMM) was evaluated in our previous study. This paper compares the protective efficacy of rBCG-AMM, rBCG-A which overexpresses Ag85B and BCG in C57BL/6 mice. There was no significant difference in proliferation characteristics among rBCG-AMM, rBCG-A and BCG. The growth characteristics of rBCG-AMM in host tissue were identical to control BCG, suggesting the improved protective efficacy was directly related to the expression of the Ag85B-Mpt64(190-198)-Mtb8.4 fusion protein. The protective experiment demonstrated that rBCG-AMM could confer similar or even better protective efficacy against Mycobacterium tuberculosis infection compared with BCG or rBCG-A as evaluated by bacterial organ loads, lung histopathology and net weight gain or loss. The results suggested that the recombinant BCG: rBCG-Ag85B-Mpt64(190-198)-Mtb8.4 is a potential vaccine candidate for further study.