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Background: Although pentavalent antimony compounds are used as antileish-manial drugs but they are associated with limitations and several adverse complica-tions. Therefore, always effort to find a new and effective treatment is desired. In this study, the effect of ZnO nanoparticles with mean particle size of 20 nanome-ters (nm) on Leishmania major promastigotes and amastigotes was evaluated. Methods: Viability percentage of promastigotes after adding different concentra-tions of ZnO nanoparticles (30, 60, 90 and 120 μg/ml) to the parasite culture was evaluated by MTT assay. In the flow cytometry study, Annexin V-FITC Apoptosis detection Kit was used to study the induced apoptosis and necrotic effects. Result : IC50 after 24 hours of incubation was 37.8 μg/ml. ZnO nanoparticles ex-ert cytotoxic effects on promastigotes of L. major through the induction of apopto-sis. A concentration of 120 μg/ml of ZnO nanoparticles induced 93.76% apoptosis in L. major after 72 hours. Conclusion: ZnO NPs can induce apoptosis in L. major by dose and time-de-pended manner in vitro condition.