Tuberculosis (TB) infection affects a quarter of the global population resulting in a large burden of TB disease and mortality. The long-term control of TB requires vaccines with greater efficacy and durability than the current Mycobacterium bovis Bacille Calmette-Guérin (BCG). Pulmonary immunization may increase and prolong immunity at the site of Mycobacterium tuberculosis infection. We have investigated recombinant influenza A viruses (rIAVs) expressing the p25 CD4 T cell epitope of M. tuberculosis Ag85B for single and sequential immunization against M. tuberculosis infection. Intranasal immunization with single dose of rIAV X31 (H3N2 strain) expressing the p25 epitope (X31-p25)， induced p25-specific CD4 T cells and conferred protection against aerosol delivery of M. tuberculosis infection in the lungs. To enhance this effect， prime-boost immunization with hetero-subtypic rIAVs was examined. Sequential immunization with X31-p25 and a second rIAV， PR8 (H1N1 strain) expressing the same epitope (PR8-p25)， increased the frequency of p25-specific IFN-γ T cell responses and polyfunctional CD4 T cells producing IFN-γ， IL-2， and TNF， compared to immunization with each rIAV alone. This combination resulted in protection against M. tuberculosis in both the lungs and spleen. Therefore， our study revealed that rIAV is not only an efficient vector to induce protective immunity in the lungs， but also has a potential use for sequential immunization with heterologous rIAV to boost the immunogenicity and improve the protection against M. tuberculosis.