Mycobacterium tuberculosis (M tuberculosis) has a penetrance of its host population.About one-third of the human population would have a positive skin test for the infection.The latest estimate of the global tuberculosis (TB) mortality among human immunodeficiency virus (HIV)-negative people fell from 30 to 20 per 100,000 population (36%) between 1990 and 2009 and could be halved by 2015.The overall decline (when including HIV-positive people, who comprise 12% of all TB cases) was 19%.Nevertheless, a rapid spread of multi-drug resistant bacteria causing about 0.5 million TB cases per year has worsened the problem during recent years.The conventional anti-TB vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), has still widely used to prevent TB and is given to millions of infants worldwide as unique vaccine since 1921.However, the protective efficacy of BCG has shown variable effects against adult pulmonary TB in different trials.There is no effective vaccine against M tuberculosis infection in view of the weakness of BCG, therefore, the development for a new TB vaccine candidate may consider it.In the study, we developed the DNA vaccine encoding fusion protein of antigen 85A and 6kDa early secretory antigen target of M tuberculosis as well as the cytokine IL-21 to investigate its immune protective efficacy against M tuberculosis challenge in mice after the DNA vaccine priming and BCG boosting.Compared with the different control groups, the intranasal (i.n.)DNA vaccine priming twice and BCG boosting once markedly increased the cytotoxicities of natural killer cells and splenocytes, enhanced the interferon-γ level in the splenocyte supernatant as well as sIgA level in bronchoalveolar lavage in the vaccinated mice.This heterologous prime-boost strategy significantly decreased the bacterial load in the mouse lungs in contrast to that of intranasal or subcutaneous BCG immunization alone.Our data demonstrated that i.n.DNA vaccine Ag85A-ESAT-6-IL-21 priming twice and i.n.BCG boosting once significantly reduced the bacterial burdens in the mice.The IL-21 is a promising immune adjuvant to enhance immunogenicity of DNA vaccine and induces powerful protective efficacy against M tuberculosis challenge in mouse model.These findings provide further approaches for mucosal targeted prime-boost vaccination to fight against tuberculosis.