Edible mushrooms are valuable source of biologically active compounds with beneficial impact on health. Their anticancer and immunomodulatory properties are mainly attributed to polysaccharides, especially their derivatives. Polysaccharide-derived oligosaccharides have been widely used as food additives, prebiotic supplements, drug delivery, immunoenhancers and animal feed. The biological activity of (1-3)- α -D-glucooligosaccharides obtained as acid-hydrolysis products of (1-3)- α -D-glucan from Laetiporus sulphureus were analyzed. Their influence on normal human colon epithelial cells (CCD 841 CoN) viability was analyzed by means of LDH assay. Anticancer properties of oligosaccharides were screened on human colon adenocarcinoma cell lines (Caco-2, LS180 and HT-29) by means of MTT and BrdU assays. Furthermore, flow cytometry was applied to cell cycle analysis. Performed studies reveled that tested compounds were not toxic to normal colon epithelial cells in whole range of investigated concentrations (0-1000µg/mL), at the same time antiproliferative effect of oligosaccharides was observed in colon cancer cells. The strongest inhibition of cells proliferation was observed on HT-29 cells (IC50 310.6 µg/mL) (MTT test). Nevertheless more specific BrdU assay reveled significant decrease of DNA synthesis only in case of LS180 cells. In order to determine mechanism by which tested compound influence cancer cell proliferation flow cytometry was applied. Performed studies demonstrated alterations in cell cycle progression only in case of HT-29 cells, where (1-3)-α-D-glucooligosaccharides at concentration 1 mg/mL induced cells accumulation in S phase. Our results indicate that oligosaccharides from (1→3)-α-D-glucan possess an anticancer potential and may provide a new chemopreventive option against colon cancer.