Tuberculosis (TB) is worldwide the most prevalent bacterial disease of humans and a new effective vaccine is urgently needed. In this study we have evaluated the vaccine potential of a Mycobacterium tuberculosis (Mtb) antigen of the PPE (Pro-Pro-Glu) protein family, namely ppe44 (Rv2770c). This family includes 69 proteins rich in glycine and together with the PE (Pro-Glu) proteins accounts for ~10% of the coding capacity of the Mtb genome. Little is known about their function but their polymorphic nature suggests that they represent antigens of immunological relevance. ppe44-specific immune responses generated by an infection with Mtb of mice were analyzed and the immunogenicity and protective efficacy of a plasmid DNA vaccine coding for ppe44 (pDNA-ppe44) and of recombinant ppe44 protein formulated in DDA adjuvant (rec-ppe44) were evaluated. The results presented show that ppe44-specific immune responses are detected in mice acutely and chronically infected with Mtb. Vaccination of C57BL/6 and BALB/c mice with pDNA-ppe44 and rec-ppe44 generates strong cellular and humoral antigen-specific immune responses. Immunodominant T cell epitopes have been mapped using overlapping synthetic peptides covering the entire ppe44 sequence and IL-2+/IFN-γ+ and IFN-γ+/cytotoxic epitopes have been identified. Most importantly, vaccination of C57BL/6 mice with pDNA-ppe44 and rec-ppe44 followed by an intratracheal challenge with virulent Mtb results in a protective efficacy comparable to the one afforded by BCG in terms of reduction in bacterial load in the lungs. Finally, vaccination of (B6D2)F1 mice with rec-ppe44 followed by an intravenous challenge with virulent Mtb results in a protective efficacy comparable to the one afforded by BCG in terms of long-term survival. Taken together these results indicate that ppe44 of Mtb is a protective antigen that could be included in novel sub-unit TB vaccines and that warrants further analysis.