New approaches consisting of ‘multistage' vaccines against (TB) are emerging that combine early antigenic proteins with latency-associated antigens. In this study, HspX was tested for its potential to elicit both short- and long-term protective immune responses. HspX is a logical component in vaccine strategies targeting protective immune responses against primary infection, as well as against reactivation of latent infection, because as previously shown, it is produced during latency, and as our studies show, it elicits protection within 30 days of infection. Recent studies have shown that the current TB vaccine, bacilli Calmette-Guerin (BCG), does not induce strong interferon-γ T-cell responses to latency-associated antigens like HspX, which may be in part why BCG fails to protect against reactivation disease. We therefore tested HspX protein alone as a prophylactic vaccine and as a boost to BCG vaccination, and found that HspX purified from M. tuberculosis cell lysates protected mice against aerosol challenge and improved the protective efficacy of BCG when used as a booster vaccine. Native HspX was highly immunogenic and protective, in a dose-dependent manner, in both short- and long-term infection models. Based on these promising findings, HspX was produced as a recombinant protein in E. coli, as this would enable facile purification; however, recombinant HspX (rHspX) alone consistently failed to protect against aerosol challenge. Incubation of rHspX with mycobacterial cell lysate and re-purification following incubation restored the capacity of the protein to confer protection. These data suggest the possibility that the native form may chaperone an immunogenic and protective antigen that is mycobacteria-specific.