Abstract: Objective: In this study, the polysaccharide of chestnut fruit wine was extracted, purified and characterized, and its anti-fatigue activity was studied. Methods: The crude polysaccharide of chestnut fruit wine was extracted by ultrasonic-assisted water extraction and alcohol precipitation, and purified and separated by ultrafiltration technology. Its structure was analyzed by ion chromatography, gel permeation chromatography, ultraviolet spectroscopy and infrared spectroscopy, and its anti-fatigue effect was studied by weight-bearing swimming test in mice. Results: CFWP-1(>100 kU) and CFWP-2 (<100 kU) were isolated and purified by ultrafiltration membrane. Both of these polysaccharides were heterogeneous, with the average molecular weight of CFWP-1 being 132351 Da and 664 Da, and the average molecular weight of CFWP-2 being 133619 Da and 664 Da. The results of monosaccharide analysis showed that CFWP-1 and CFWP-2 were mainly composed of arabinose, galactose, glucose, galacturonic acid and mannouronic acid, and the glucose content in these two polysaccharides was more than 98%, which belonged to neutral glucan. UV spectra showed that the purity of the two polysaccharides was high. Infrared spectroscopy analysis showed that both polysaccharides had typical absorption peaks of polysaccharides. Anti-fatigue experiments showed that both CFWP-1 and CFWP-2 could significantly increase the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum of mice (P<0.05). The levels of lactate dehydrogenase (LDH), serum urea nitrogen (BUN), blood lactate (BLA), malondialdehyde (MDA) and creatine kinase (CK) were significantly decreased (P<0.05), and the glycogen storage level was significantly increased. It had a good protective effect on skeletal muscle in mice. The effect of CFWP-2 was better than that of CFWP-1 in a dose-dependent manner. Conclusion: The polysaccharide extracted from chestnut fruit wine would had good anti-fatigue effect.