Understanding functional interactions between DCs and antigens is necessary for achieving an optimal and desired immune response during vaccine development. Here, we identified and characterized protein Rv2299c (heat-shock protein 90 family), which effectively induced DC maturation. The Rv2299c-maturated DCs showed increased expression of surface molecules and production of proinflammatory cytokines. Rv2299c induced these effects by binding to TLR4 and stimulating the downstream MyD88-, MAPK- and NF-κB-dependent signaling pathways. The Rv2299c-maturated DCs also showed an induced Th1 cell response with bactericidal activity and expansion of effector/memory T cells. The Rv2299c-ESAT-6 fused protein had greater immunoreactivity than ESAT-6. Furthermore, boosting BCG with the fused protein significantly reduced hypervirulent Mycobacterium tuberculosis HN878 burdens post-challenge. The pathological study of the lung from the challenged mice assured the efficacy of the fused protein. The fused protein boosting also induced Rv2299c-ESAT-6-specific multifunctional CD4+ T-cell response in the lungs of the challenged mice. Our findings suggest that Rv2299c is an excellent candidate for the rational design of an effective multiantigenic TB vaccine.