To improve DNA vaccination against Mycobacterium tuberculosis, we evaluated the effectiveness of a Sindbis virus-based DNA construct expressing the tuberculosis antigen 85B (Sin85B). The protective efficacy of Sin85B was initially assessed by aerogenically challenging immunized C57BL/6 mice with virulent Mycobacterium tuberculosis. At 1 and 7 months postinfection, the lung bacterial burdens were considerably reduced and the lung pathology was improved in vaccinated mice compared to naive controls. Furthermore, the mean survival period for Sin85B-immunized mice (305 ± 9 days) after the tuberculous challenge was extended 102 days relative to the naive mice (203 ± 13 days) and was essentially equivalent to the survival time of Mycobacterium bovis BCG-vaccinated mice (294 ± 15 days). The essential role of gamma interferon (IFN-γ) in Sin85B-mediated protection was established by showing that significantly increased levels of IFN-γ mRNA were present postinfection in lung cells from vaccinated mice relative to control mice and by demonstrating that IFN-γ depletion prior to challenge abolished the vaccine-induced protection. The substantial antituberculosis protective responses induced by Sin85B immunization of CD4^−/− mice strongly suggested that CD8 cells partially mediate Sin85B-induced protective immunity. Interestingly, Sin85B vaccination did not protect RNase L^−/− (a key enzyme in the innate antiviral response) mice while significant protection was detected in RNase L^−/− mice immunized with either BCG or a conventional DNA plasmid expressing antigen 85B. These data show that immunization with Sin85B offers protection similar to BCG in a murine model of pulmonary tuberculosis and suggest that Sin85B-induced protection is dependent upon both innate and acquired immune mechanisms.