The immunogenicity and protective efficacy of combined DNA priming, Bacillus Calmeette Guerin(BCG) boosting vaccination in mice were examined. Following intravenous challenge with virulent M. tuberculosis H37Rv, the BCG boost approach resulted in significant protection in both lungs (1.3, P < 0.01) and spleens (1.1, P < 0.01) compared with the saline control. In addition, this approach also have much better protection than that vaccination with combined DNA or BCG alone (P < 0.05). The elevated CD4⁺ and CD8⁺ T cells percentage (P < 0.05) in PBMC and higher IFN-γ concentration (1250 ng/L, P < 0.01), IL-2 concentration (230 ng/L, P < 0.05) stimulated by Ag85B protein in spleen cells supernatant in BCG boosted mice 21 days after third vaccination, indicating that the DNA prime-BCG boost strategy significantly enhanced the Th1 type cell response, which is correlated with the protective efficacy against tuberculosis. Furthermore, immuno-histochemistry assay showed that there were more Perforin expression cells, secreted mainly by CD8⁺ T cells, in the lung tissue of the mice primed with DNA prior to BCG. Taken together, the heterogonous boost combination provided superior protection by stronger CD4⁺ (Th1) and CD8⁺T cell mediated immune response, which suggested that it will be a promising regimen for MTB vaccine development.