Background: The present study aimed to survey antileishmanial activity of methanolic Holothuria leucospilota extract against Leishmania major promastigotes in vitro. Methods: Promastigotes were cultured in RPMI 1640 and after reaching the stationary phase, the study was conducted with different concentrations of the extract. Afterwards, MTT colorimetric assay for the obtaining of 50% inhibitory concentration (IC 50 ) was utilized. Furthermore, in order to determine the possible induction of apoptosis in L. major promastigotes, flow cytometry and DNA fragmentation methods were employed using annexin-V FLUOS staining kit and DNA ladder kit, respectively. Results: The IC 50 value of H. leucospilota extract at three time points of 24, 48, and 72 h was estimated 2000, 300 and 85 µg/ml, respectively. In addition, the extract revealed a dose and time-dependent antileishmanial activity. Furthermore, various characteristics of apoptosis appeared after L. major promastigotes treatment, which included cell shrinkage, formation of apoptotic bodies, blebbing of the cell membrane, and externalization of phosphatidylserine, although no laddering pattern was observed. Conclusion: The methanolic extract of H. leucospilota possesses lethal effect on L. major promastigotes and induces the apoptosis in parasites. Further studies are required to address the apoptosis mechanism in vivo.