The previous research of our group found that the petroleum ether-soluble portion of the ethanol extract of Cirsium setosum had a good inhibitory effects on cytotoxic activity of a variety of human tumor cells in vitro. Now it is necessary to study the chemical composition of unresearched strong active subfractions， and to explore the functional factors that exert cytotoxic activities. Under the guidance of inhibiting cytotoxic activity in vitro， various chromatographic techniques were used for separation and purification， and spectroscopy methods were used for structural elucidation， and the structures were determined by comparison with known literature data. Five steroids were isolated from an active subfraction of the petroleum ether of ethanolic extract of Cirsium setosum， and determined as 3β-hydroxystigmast-5-en-7-one， stigmast-22-ene-3β，5α，6β-triol， 6β-hydroxy- stigmasta-4-en-3-one， 6β-hydroxy-stigmasta-4，22-dien-3-one and 6β-hydroxycampest-4-en-3-one. To the best of our knowledge， this is the first report showing the presence of all the compounds in the genus Cirsium. In the in vitro assays， except for the stigmast-22-ene-3β，5α，6β-triol， the other four compounds all showed selective cytotoxicities.