An effective Leishmania vaccine must induce strong Th1 immune responses and generate long-term effectiveness in the host. To date, most designated anti-Leishmania vaccines have shown poor efficacy in clinical studies. One of the most effective approaches in enhancing the efficacy of vaccines to generate protective immunity is the use of multiple distinct antigens in a single vaccination regimen. For this reason, we investigated the immunogenicity and protective efficacy of liposomal formulation composed of DOTAP:Cholesterol, protamine and CpG ODNs (LPD NPs) containing rLmaC1N and rgp63 recombinant proteins (LPD-rLmaC1N/rgp63) as an effective vaccine. Then, we have investigated the immunogenicity and protective efficacy of LPD-rLmaC1N/rgp63 in experimental animal model of cutaneous leishmaniasis compared to free antigens. Our study showed that mice immunization with LPD-rLmaC1N/rgp63 significantly activates Th1 immune response and enhances IFN-γ production and IgG2a levels. Notably, LPD-rLmaC1N/rgp63 immunization greatly reduced the number of live parasites in the spleen and footpads of immunized mice compared to other vaccinated groups. As well, the thickness of footpad was remarkably smaller in mice immunized with LPD-rLmaC1N/rgp63. In conclusion, our study indicated that LPD-rLmaC1N/rgp63 could be regarded as a promising multiple-antigen candidate vaccine compared to single antigen counterparts to generate protective immunity against leishmaniasis. The potential advantages of this formulation may warrant further investigation.