The development of active polysaccharides from edible mushrooms for antitumor drugs or functional foods has become a research hotspot. In this study, the physicochemical properties and monosaccharide composition of four homogeneous polysaccharides (CASP-0, CASP-1, CASP-2 and CASP-3) purified from alkali-extracted Stropharia rugosoannulata polysaccharides by DEAE-52 column were investigated, and the inhibitory effect against colon cancer HT-29 cell in vitro were explored. Results showed that four polysaccharides were homogeneous with molecular weights of 84.8 KDa (CASP-0), 11.1 KDa (CASP-1), 58.6 KDa (CASP-2) and 97.8 KDa (CASP-3). CASP-0 and CASP-3 contained glucose, mannose and galactose in molar ratios of 83.76:13.96:2.27 and 89.72:4.68:5.59, while CASP-1 and CASP-2 contained glucan. The four polysaccharides significantly reduced the cell viability of HT-29 cell in dose-dependent and time-dependent manner. With the increase of polysaccharide concentration, CASPs significantly inhibited the migration and invasion of HT-29 cell. The four polysaccharides induced HT-29 cell apoptosis by up-regulating the expression of Caspase-3 and Bax protein, and down-regulating the expression of Bcl-2 protein, suggesting that HT-29 cell apoptosis may be a mitochondria-mediated pathway. This study provides a theoretical basis for the potential application in medicine and functional food.