Objectives: The medicinal plant of Ornithogalum cuspidatum (Liliaceae) uses widely in Iranian traditional medicine for treatment of respiratory and inflammatory disease. Previous study on methanolic extract of Ornithogalum cuspidatum showed that it has cytotoxic effect. Therefore we aimed to study the cytotoxic effect of methanolic extract fractions of Ornithogalum cuspidatum on WEHI-164, fibrosarcoma cancer cell line.Methods: Fractions of methanolic extract of Ornithogalum Cuspidatum was prepared using SPE (Solid Phase Extraction ).WEHI-164 cancer cell line were treated with different concentrations of 10%, 20%, 40%, 60%, 80% and 100% fractions of methanolic extract of Ornithogalum cuspidatum for 12 and 24 hours. Cytotoxicity and cell viability were measured by MTT assay. ELISA was performed to determine apoptosis after 24 hours in different concentrations of methanolic fractions.Results: 10% and 20% fractions of methanolic extract had not cytotoxic effect but 40%, 60%, 80% and 100% fractions induced a significant growth inhibition in WEHI-164 cancer cell line with LC50 of 200, 96, 76 and 73 mg/ml respectively. Morphological changes of cell death in treated WEHI-164 cells were mediated via apoptosis. Apoptosis in WEHI-164 cells after 24 hours treatment with 200 mg/ml of 40%, 60%, 80% and 100% fractions were 36, 41, 42 and 49% respectively.Conclusion: The results of this study suggest that 80% and 100% fractions of methanolic extract of Ornithogalum cuspidatum contain higher cytotoxic and apoptotic compounds on WEHI-164 cancer cells and can be candidate for identifying and purifying of cytotoxic bioactive compounds.