Six water-soluble polysaccharides (S-WPLE-I-a, S-WPLE-I-b, S-WPLE-II-a, S-WPLE-II-b, S-WPLE-III-a and S-WPLE-III-b) were obtained from Lentinus edodes and purified by gel-permeation chromatography (GPC). The fractions were analyzed for monosaccharide composition, molecular weight, and then tested for cytotoxicity activity. The results from high performance liquid chromatography (HPLC) revealed that the water-soluble fractions were heteroglucan containing mainly glucose (Glc), galactose (Gal) and mannose (Man) at various ratios. All the polysaccharide fractions exhibited antitumor activities against Sarcoma 180 (S-180) solid tumor cells and human colorectal cancer cell lines (HT-29 and HCT-116) in vitro at the dose of 5mg/ml. The antitumor activities of the polysaccharides were related to their monosaccharide content and molecular weight. The effects of Gal, Man and bound protein on the improvement of antitumor activities of polysaccharides might not be negligible. The results also revealed that there was selectively higher antitumor activity of the polysaccharides against suspended cells (S-180) than adherent ones (HT-29 and HCT-116).