OBJECTIVES: To design a multivalent DNA vaccine encoding the most immunogenic regions of the antigens including TSA (Thiol-specific antioxidant protein), LmSTI1 ( stress-inducible protein1), LACK ( homologue of receptors for activated C Kinase), and KMP11 (kinetoplastid membrane protein-11) on BALB/c mice. MATERIALS AND METHODS: The chimeric construct was generated including the most immunogenic epitopes containing a combination of domains and oligopeptides of the aforementioned genes. The construct was cloned into pcDNA 3.1 plasmid and named "pleish-dom." Following intramuscular injection of mice, the capability of the vector pleish-dom alone and with pIL-12 (expressing murine IL-12) to raise protective cytokines and parasite burden was evaluated in the BALB/c mice as a susceptible animal model against . RESULTS: The immunized mice with pleish-dom/pIL-12 showed the highest and the lowest levels of interferon-gamma (IFN-γ) and interleukin-10 (IL-10), as well as the lowest leishmanin skin test (LST) reactions, were found through 8 weeks post-infection. CONCLUSION: Although the obtained DNA vaccine from the immunogenic chimeric protein of antigens was able to induce a high level of IFN-γ, it partially protected mice against L. major. However co-administration with pIL-12 led to shift immune response to Th1 phenotype, granuloma formation, and lowered parasite burden, and consequently distinct protection was found.