Growing evidence shows that antioxidant proteins of Leishmania could be used as vaccine candidates. In this study, we report the efficacy of Leishmania donovani iron superoxide dismutase B1 (LdFeSODB1) as a vaccine antigen in BALB/c mice in a DNA-protein prime-boost immunization regimen in the presence or absence of murine granulocyte macrophage colony stimulating factor (mGMCSF) DNA adjuvant. The expression study confirmed that LdFeSODB1 is expressed in mammalian cells and mGMCSF fusion mediates the secretion of the recombinant protein. Heterologous immunization with LdFeSODB1 induced a strong antibody- and cell-mediated immune response in mice. Immunization triggered a mixed Th1/Th2 response as evidenced by the ratio of IgG2a to IgG1. Antigen-stimulated spleen cells from the immunized mice produced high level IFN-γ. Multiparametric flow cytometry data showed that immunization with LdFeSODB1 induced significantly higher expression of TNF-α or IL-2 by antigen-stimulated T cells. Eight weeks after L. major infection, immunization with the antigen shifted the immune response to a more Th1 type than the controls as demonstrated by IgG2a/IgG1 ratio. Moreover, IFN-γ production by antigen-stimulated spleen cells from immunized mice remained high. The footpad swelling experiment showed that immunization with LdFeSODB1 resulted in partial protection of mice from a high dose L. major infection.