We examined the protective effect of autoclaved Leishmania major (ALM) vaccine in combination of either rectal or subcutaneous BCG on susceptible BALB/c mice. One month after BCG vaccination, BALB/c mice were immunized subcutaneously twice with ALM + alum at 3 weeks intervals. Three weeks after booster injection, 5 × 10(5) stationary phase L. major promastigotes were inoculated subcutaneously in one footpad. Immunological evaluation at before and post infectious challenge showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. High level of interferon gamma was induced in the spleen, and significant increase in the serum ratio of IgG2a/IgG1 was observed only in rectal immunized group. Rectal immunized mice showed comparable nitric oxide production and iNOS induction in peritoneal macrophages (P ≤ 0.05). The obtained results in rectal BCG vaccinated group showed no mortality but low parasite burden in the liver and spleen. In conclusion, the results of our study indicated that co-administration of rectal BCG and ALM induced protective type 1 immune responses against L. major infection. This safe and effective mucosal vaccine could be useful in prevention of human leishmaniasis infections.