An efficacious tuberculosis (TB) vaccine will probably need to induce both CD4 and CD8 T-cell responses specific to a protective Mycobacterium tuberculosis antigen(s). To achieve this broad cellular immune response we tested a heterologous DNA/protein combination vaccine strategy. We used a purified recombinant protein preparation of a unique M. tuberculosis antigen (rMT1721) found in the urine of TB patients, an optimized plasmid DNA expressing this protein (DNA-MT1721), and a Toll-like receptor 4 agonist adjuvant. We found that priming mice with DNA-MT1721 and subsequently boosting with rMT1721 elicited high titres of specific IgG1 and IgG2a antibodies as well as high magnitude and polyfunctional CD4+ T-cell responses. However, no detectable CD8+ T-cell response was observed using this regimen of immunization. In contrast, both CD4+ and CD8+ T-cell responses were detected after a prime/boost vaccination regimen using rMT1721 as the priming antigen and DNA-MT1721 as the boosting immunogen. These findings support the exploration of heterologous DNA/protein immunization strategies in vaccine development against TB and possibly other infectious diseases.