Objective: Although Iron is a crucial element for many metabolic pathways of the body, the excess iron may induce apoptosis in some cell types such as macrophages. In the present investigation, the role of iron overload in inducing apoptosis of Balb/c mice macrophages infected with L. major in vitro, as a selective model, was studied. Materials and Methods: The peritoneal macrophages were harvested and cultured with different concentrations of iron to refuse its cytotoxic effect. Then, the macrophages were harvested and cultured with or without Leishmania in the presence of iron or donated reagent [S-Nitro-N-Acetylpenicillamine(SNAP)] or an inhibitor of NO, synthase [NG-Methyl-L-Arginine(NMMA)]. The concentration of NO as an immunological mediator in culture supernatants was measured after 18 hour incubation. Simultaneously, macrophages undergoing apoptosis were identified by fluorescence and electron microscopy. Results: The findings showed that there is a statistically significant relationship between iron overload and apoptosis (p<0.05). Apoptosis rate had also increased in the macrophages cultured with iron, SNAP, NMMA, as compared with control group. Conclusion: Iron influences the apoptosis rate and NO production in the macrophages infected with L. major.