Background: Pentavalent antimonate compounds are the routine drugs for leishmaniasis. Apart from side effects, relapse and drug resistance are among the main problems of these compounds. Hawthorn has antioxidant and antibacterial effects. The aim of present study was to determine the anti-leishmanial effect of hawthorn fruit extract in vitro. Materials and Methods: In this experimental study different doses of hawthorn fruit extract was added to Leishmania major promastigotes. Cytotoxicity was investigated by both direct counting and MTT assay after 24, 48 and 72 hours. In addition, hawthorn extract was added to amastigote-infected macrophages and then the mean of amastigote per macrophage was counted after 24 and 48 hours. Data were analyzed using one-way ANOVA. Results: The cytotoxicity of the highest and lowest concentration of the extract (60 µg/ml and 0.5 µg/ml) after 24, 48 and 72 hours was 31.86, 27.93; 20.11, 20.94; and 35.19, 55.14, respectively. After 24 hours the IC50 was 49.13 μg/ml; the mean number of amastigote per macrophage in Control and Plant extract-treated groups (1, 10 and 100 μg/ml) were: 3.91; (1.97, 1.76 and 1.78), respectively. Conclusion: The results showed that hawthorn fruit extract has no significant cytotoxic effect on promastigotes. However, to some extent it can inhibit the proliferation of amastiogtes inside macrophages