Background: The current study aimed to investigate the possible cross-protective effects of attenuated L. major against L. infantum in BALB/c mice. Methods: This experimental study was performed in 2017 in Shiraz University of Medical Sciences, Shiraz, Iran. The attenuated strain of L. major was prepared by continuous weekly subculturing of the parasite. Forty-eight female BALB/c mice were divided into eight groups. Group 1 injected (ID) with wild type of L. major; group 2 injected (IV) with L. infantum; group 3 injected (ID) with attenuated L. major; group 4 injected (ID) with attenuated L. major, and after three weeks challenged (IV) with L. infantum; group 5 injected (IP) with attenuated L. major; group 6 injected (IP) with attenuated L. major, and challenged (IV) with L. infantum (IV); group 7 injected (IV) with attenuated L. major; and finally group 8 injected (IV) with attenuated L. major and after three weeks challenged (IV) with L. infantum. Forty-five days post-infection, the parasite load in the spleen and liver of the mice was determined as Leishman-Donovan units (LDU). Results: The differences in mean of LDU of spleen between different groups were statistically significant (P<0.048). In addition, the differences in percent of infection in liver between pairwise comparisons of groups were statistically significant (P<0.05). The highest intensity of infection was observed in group 2 while low intensity of infection was seen in groups 3, 4 and 5. Conclusion: Live attenuated L. major can induce substantial protection against L. infantum, particularly when the parasites were injected intravenously.