Background To date, no universally effective and safe vaccine has been developed for general human use. Leishmania donovani Peroxidoxin-1 (LdPxn-1) is a member of the antioxidant family of proteins and is predominantly expressed in the amastigote stage of the parasite. The aim of this study was to evaluate the immunogenicity and protective efficacy of LdPxn-1 in BALB/c mice in heterologous DNA-Protein immunization regimen in the presence of fusion murine granulocyte-macrophage colony-stimulating factor (mGMCSF) DNA adjuvant. Methodology and Principal Findings A fusion DNA of LdPxn1 and mGMCSF was cloned into a modified pcDNA vector. To confirm the expression in mammalian system, Chinese hamster ovary cells were transfected with the plasmid vector containing LdPxn1 gene. BALB/c mice were immunized twice with pcDNA-mGMCSF-LdPxn-1 or pcDNA-LdPxn1 DNA and boosted once with recombinant LdPxn-1 protein. Three weeks after the last immunization, mice were infected with Leishmania major promastigotes. The result showed that immunization with pcDNA-mGMCSF-LdPxn1 elicited a mixed Th-1/Th-2 immune response with significantly higher production of IFN-γ than controls. Intracellular cytokine staining of antigen-stimulated spleen cells showed that immunization with this antigen elicited significantly higher proportion of CD4+ T cells that express IFN-γ, TNF-α, or IL-2. The antigen also induced significantly higher proportion of multipotent CD4+ cells that simultaneously express the three Th-1 cytokines. Moreover, a significant reduction in the footpad swelling was seen in mice immunized with pcDNA-mGMCSF-LdPxn1 antigen. Expression study in CHO cells demonstrated that pcDNA-mGMCSF-LdPxn-1 was expressed in mammalian system. Conclusion The result demonstrates that immunization of BALB/c mice with a plasmid expressing LdPxn1 in the presence of mGMCSF adjuvant elicits a strong specific immune response with high level induction of multipotent CD4+ cells that mediate protection of the mice from Leishmania major infection. To our knowledge, this is the first study showing the vaccine potential of Leishmania peroxidoxin -1., Author Summary Leishmaniasis, a disease caused by protozoan parasites under the genus Leishmania, claims the lives of thousands of people annually. It mainly affects people in poor communities in Africa, Asia and South America. Although several drugs are available for the treatment of leishmaniasis, their efficacy is limited by the emergence of drug resistant parasite strains and by the inherent side-effects of some drugs. In light of these challenges, developing effective vaccine is considered as a crucial step in the control and ultimate elimination of the disease. In this study, we have evaluated the potential of the antioxidant Leishmania Peroxidoxin-1 as a candidate vaccine for leishmaniasis. The efficacy of the candidate vaccine was assessed in DNA-Protein immunization strategy in mice. We also investigated the adjuvant role of GMCSF DNA fused with the vaccine antigen in a pcDNA plasmid vector. The result showed that Leishmania Peroxidoxin-1 together with fusion GMCSF adjuvant in a pcDNA plasmid induces a partially protective immune response in mice. Further analysis of the immune response demonstrated that the antigen-adjuvant combination elicits CD4+ T cells that express IFN-γ, TNF-α, or IL-2. The antigen also induced a high frequency of CD4+ T cells that simultaneously express all the three cytokines. The study on samples taken from leishmaniasis patients showed that the recombinant Leishmania Peroxidoxin-1 protein is recognized by and elicit immune response in humans, a crucial requirement in the development of a vaccine.