Background: Cutaneous leishmaniasis is a common parasitic disease in Iran being mainly caused by Leishmania (L.) major. The aim of this study was to investigate the occurrence of apoptosis in the spleen and liver of female mice infected with L. major.Methods: BALB/c mice were randomly assigned into the control and experimental groups (ten mice per group). The experimental groups were subcutaneously inoculated with promastigotes of L. major at stationary phase. The animals were sacrificed after 20, 40, 60, 90, and 120 days of injection. The liver and spleen were analyzed for various parameters of apoptosis.Results: Activities of superoxide dismutase and caspase-3, levels of superoxide anion production and malondialdehyde, and the percent of DNA fragmentation were increased in the liver and spleen of the infected mice. Catalase activity in the liver was increased, while glutathione level in both tissues was decreased after 90 and 120 days of infection. The numbers of apoptotic nuclei in the spleen were higher than the liver at 90 and 120 days post-infection using the TUNEL method.Conclusion: L. major infection induces a time-dependent increase in apoptosis in the liver and spleen as evidenced by the production of ROS, increasing activation of caspase-3, elevated DNA fragmentation, and increasing lipid peroxidation. Induction of oxidative stress was observed in the liver and spleen after 90 and 120 days of initiation of the infection. However, the spleen tissue appears to be more sensitive to the infection to L. major on oxidative stress and apoptosis induction compared with the liver tissue.