Among several unmet needs, the identification of vaccine target antigens (Ags) that can induce powerful Ag-specific Th1 immunity is the first step toward tuberculosis vaccine development. Here, we identified MTBK20640 of Mycobacterium tuberculosis (Mtb) through comparative genomic approaches, highly conserved PE family protein in Mtb Beijing genotype, as a vaccine target and molecular characterization was performed by its interaction with dendritic cells (DCs). MTBK20640-stimulated DCs displays increased expression of surface molecules and secretion of proinflammatory cytokines. With the ex vivo stimulation of MTBK20640 induces IFN-γ production to a similar extent as those stimulated with ESAT-6 Ag in lung and spleen cells from Mtb-infected mice. Based on these immunostimulatory properties, this Ag candidate was evaluated as prophylactics. Prior to challenge, mice immunized with MTBK20640 adjuvanted with GLA-SE exhibited Ag-specific IFN-γ secretion in spleen and lung cells. Also, expansion of CD4+/CD8+ effector/memory T cells was observed in spleen and lung in MTBK20640-immunized group. Following challenge with hyper-virulent Mtb K strain, MTBK20640/GLA-SE caused a significant reduction in bacteria and reduced lung inflammation. Importantly, the protective efficacy of the MTBK20640 subunit vaccine was correlated with remarkable generation of Ag-specific CD4+ T cells co-producing IFN-γ, TNF-α, and IL-2. These results suggest that MTBK20640 possesses a Th1-polarizing ability and promotes protective immunity against Mtb infection by sustaining high levels of multifunctional T cells. We are currently under investigation whether this conserved antigen has potential as a novel vaccine candidate against the hyper-virulent Mtb HN878 strain.