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Objective: To clarify the structural characteristics of selenium polysaccharides from Morchella esculenta, and their ameliorative effects on exercise-fatigued rats. Methods: Polysaccharides from Morchella esculenta were extracted using ultrasound-assisted hot water extraction. Purified polysaccharides (Msp-1) were obtained through DEAE cellulose column chromatography and Sephadex G-100 column chromatography. Nitric acid-sodium selenite method was used to prepare selenized polysaccharide (Se-Msp1) from purified polysaccharides. The structures of Msp-1 and Se-Msp1 were characterized. The swimming time of rats was measured by exhaustive swimming test, and the weighted swimming model was established at the same time. The rats were randomly divided into quiet control group, exercise fatigue control group, positive control group (salidroside, 100 mg/kg), normal polysaccharide group (Msp-1, 100 mg/kg), and selenized polysaccharide low, medium, and high dose groups (Se-Msp1, 50, 100, and 200 mg/kg, respectively). The rats were orally administered with 0.1 mL/(10 g·bw) for 5 weeks. The content of blood lactic acid (BLA), blood urea nitrogen (BUN), hepatic glycogen (HG), and muscular glycogen (MG), as well as malondialdehyde (MDA) content, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities in skeletal muscle mitochondria, were measured to evaluate the anti-exercise fatigue effect of Se-Msp1. Results: The major component of the purified polysaccharides from Morchella esculenta was Msp-1, which accounted for 85.25%. Infrared spectroscopy showed that the heteromeric carbon of Se-Msp1 was α configuration, and characteristic absorption peaks of selenium were observed, indicating that Se-Msp1 was successfully selenided. The Se content in Se-Msp1 was 9.56 mg/g, the polysaccharide content was 56.34%, with uronic acid content of 36.82%, and the weight average molecular weight was 3.482×105 Da, particle size of 384.71 nm, zeta potential was −45.78 mV, indicating that Se-Msp1 had a high selenium content and good stability. The monosaccharide composition of Se-Msp1 consisted of mannose, glucose, galactose, galacturonic acid, and rhamnose, with molar ratios of 1:0.42:3.57:3.34:1.86. The anti fatigue results of rats showed that compared with the control group (Con), both Msp-1 and Se-Msp1 significantly increased the exhausted swimming time (P