Abstract: Crude polysaccharides in chestnut kernel from Yanshan area were extracted by high-pressure solvent extraction. In terms of the extraction yield of polysaccharide, single factor tests and Box-Behnken design-response surface method were applied to optimize the extraction conditions. The crude polysaccharides extracted under the optimum conditions was purified through deproteinization, depigmentation and dialysis to obtain purified polysaccharides, and then the high molecular weight component (YCP-H) was separated by the preparative high-performance size-exclusion chromatography. The morphological characteristics, structural features and monosaccharide composition of YCP-H were analyzed, and a preliminary assay on in vitro antitumor activity against eight selected cancer cell lines was also carried out. The results showed that the optimum extraction conditions for crude polysaccharides were extraction temperature of 60℃, static extraction time of 9 min and extraction pressure of 6 MPa when the amount of raw materials was 10 g and cycle times was 3, with a corresponding maximum extraction yield of 19.78%±0.27%. The component YCP-H was an acidic polysaccharide which had β-pyranose configuration. YCP-H had weight-average molecular weight covering the range of 217~5900 kDa, and exhibited fiber-like morphology under scanning electron microscope. The monosaccharide composition analysis showed that it was composed of arabinose, galactose, glucose, xylose, mannose and fucose. YCP-H had significant anti-proliferative effects on human hepatoma HepG2 cells, with a half-maximal inhibitory concentration of 0.08 μg/mL. In addition, YCP-H could also effectively inhibit the proliferation of HCT-116 human colon cancer cell line and A-549 adenocarcinoma epithelial cell line.