A series of column chromatography methods including solvent extraction,Sephadex LH-20 column chromatography,silica column chromatography and HPLC methods were used to isolate and refine compounds from sponge Phyllospongia sp. from the South China Sea.The structures were identified by spectroscopic analysis and comparison with the literature.From the petroleum ether and dichloromethane soluble extract of Phyllospongia sp.,eight steroids were isolated for the first time and their structures were determined as cholesta-5,7-dien-3β-ol（1）,ergosta-5,7,24（28）-trien-3β-ol（2）,ergosta-5,7-dien-3β-ol（3）,（24E）-stigmasta-5,7,24（28）-trien-3β-ol（4）,stigmasta-5,7-dien-3β-ol（5）,5α,6α-epoxystigmasta-7,22-dien-3β-ol（6）,5α,6α-epoxycholest-7,22-dien-3β-ol（7）,5α,6α-epoxycholest-7-en-3β-ol（8）.The cytotoxic activity of all compounds against MCF-7 human breast cancer cells,HT-29 human colon cancer cells and HEP-2 human laryngeal cancer cells were tested.Compounds 1,2,7 and 8 showed significant cytotoxic activity against MCF-7 human breast cancer cells with IC50 values of 8.8,10.3,and 3.9 μmol/L respectively.