Induction of local (pulmonary) immunity plays a critical role in preventing dissemination ofMycobacterium tuberculosis (M. tb)during the early infection stage. To induce specific mucosal immunity, chitosan, a natural cationic polysaccharide, was employed as a mucosal gene carrier and complexed with pHSP65pep, our previously constructed multi-epitope gene vaccine, which induces splenic gamma-interferon (IFN-γ)+ T helper cell 1 responses. The resultant chitosan-pHSP65pep was administered intranasally to BALB/c mice with four doses of 50 μg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that the chitosan formulation significantly induced production of secretory immunoglobulin A (P< 0.05) as determined by measuring its concentrations in lung lavage fluid and enhanced pulmonary CD4+ and CD8+IFN-γ+ T cell responses (P< 0.001) compared with naked gene vaccine. Improved protection againstMycobacterium bovisbacillus Calmette-Guérin (BCG) challenge was consistently achieved by the chitosan-DNA formulation both as the vaccine alone or in a BCG prime-vaccine boost immunization scenario. Our study shows that mucosal delivery of gene vaccine in a chitosan formulation remarkably enhances specific SIgA concentrations and mucosal IFN-γ+ T cell response, which correlated positively with immunological protection.