Mycobacterium tuberculosis (M. tuberculosis) has once again become a major public health threat owing to the combined effects of a worldwide anti-tuberculosis drug resistance and the emergence of the human immunodeficiency virus pandemic. The Bacille Calmette-Guérin (BCG)-based vaccine has displayed inconsistent efficacy in different trials although it continues to be used to prevent tuberculosis in many countries. In current work, we have developed DNA vaccines expressing M. tuberculosis antigen 85A (Ag85A) and cytokine granulocyte macrophage colony stimulating factor (GM-CSF) and aimed to investigate the immune effect in mice based on BCG priming and DNA vaccine boosting and immune protection against M. tuberculosis challenge. Our results showed that the activity of cytotoxic T lymphocyte and spleen cell proliferative responses to Ag85A and IFN-gamma level as well as the specific antibody titer against Ag85A were significantly increased in mice immunized with prime-boost strategy in comparison with the mice immunized with BCG or DNA vaccine expressing Ag85A and GM-CSF alone. Meanwhile, the immune strategy of BCG-prime and DNA vaccine boost induced mice to generate efficient immune protection against M. tuberculosis challenge. Our data demonstrate that BCG-prime and DNA vaccine expressing Ag85A and GM-CSF boost provides a rational strategy for further development of DNA vaccine against M. tuberculosis infection.