The incidence of infections with nontuberculous mycobacteria (NTM) has been increasing worldwide. The emergence of multidrug-resistant NTM is a serious clinical concern, and a vaccine for NTM has not yet been developed. We previously developed a new recombinant Bacillus Calmette-Guérin (rBCG) vaccine encoding the antigen 85B (Ag85B) protein of Mycobacterium kansasii-termed rBCG-Mkan85B-which was used together with a booster immunization with plasmid DNA expressing the same M. kansasii Ag85B gene (DNA-Mkan85B). We reported that rBCG-Mkan85B/DNA-Mkan85B prime-boost immunization elicited various NTM strain-specific CD4+ and CD8+ T cells and induced Mycobacterium tuberculosis-specific immunity. In this study, to investigate the protective effect against M. kansasii infection, we challenged mice vaccinated with a rBCG-Mkan85B or rBCG-Mkan85B/DNA-Mkan85B prime-boost strategy with virulent M. kansasii. Although BCG and rBCG-Mkan85B immunization each suppressed the growth of M. kansasii in the mouse lungs, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination reduced the bacterial burden more significantly. Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination induced antigen-specific CD4+ and CD8+ T cells. Our data suggest that rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination effectively enhances antigen-specific T cells. Our novel rBCG could be a potential alternative to clinical BCG for preventing various NTM infections.