The present study evaluates the potential of improved third-generation lentivirus vector with respect to their use as an in vivo-administered T-cell vaccine against tuberculosis. Intratracheal administration of the lentivirus vector encoding MPT51 of Mycobacterium tuberculosis could induce MPT51-specific CD8+ T cells in the mediastinal lymph nodes 2 weeks after the administration. The vaccination could generate MPT51-specific memory CD8+ T cells in the lung, but not in the lymph nodes. Further, a single intratracheal immunization of MPT51 lentiviral vaccine decreased significantly the number of virulent M. tuberculosis in the lung after intratracheal challenge of the bacillus. These findings suggest that intratracheal immunization of the third-generation lentiviral vaccines is a promising vaccination strategy against pulmonary tuberculosis.