Cell-mediated immune (CMI) responses are crucial in the protection against tuberculosis. 6-kDa early secretary antigenic target (ESAT-6) is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. Flt3 ligand (FL) is a growth factor of dendritic cell (DC), which, as a powerful antigen presenting cell (APC), is essential for CMI response. In this study, we constructed a recombinant DNA vaccine encoding ESAT-6 and FL. The recombinants were identified by restriction enzyme digestion and sequence analysis. Subsequently, the recombinants were transfected into glomerular mesangial cells (GMCs) of rat. The expressed proteins were detected by Western blot. C57BL/6 female mice were immunized three times with the recombinant plasmids. The results showed that immunization with pIRES-ESAT-6 plasmid induced an obvious T-cell response compared with controls (mice immunized with PBS, pIRES or BCG). However, mice immunized with pIRES-ESAT-6-FL presented a more stronger T helper 1 (Th1)-biased response, accompanied by higher levels of lymphocytes proliferation, elevated production of Th1 cytokines (IFN-gamma and IL-2) by spleen cells, as well as increased specific antibody in sera, together with lower levels of Th2 cytokines (IL-4 and IL-10). Our results suggested that EAST-6 was a useful vaccine candidate and FL might be a powerful adjuvant, which could effectively promote T cell-mediated immune response.