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The aim of this work was to characterize spent mushroom substrate polysaccharides (MSP) from Pleurotus eryngii and their antioxidant and organ protective effects in streptozotocin- (STZ-) induced diabetic mice. The enzymatic-, acidic-, and alkalic- (En-, Ac-, and Al-) MSP were extracted from P. eryngii with snailase (4%), hydrochloric acid (1 mol/l), and sodium hydroxide (1 mol/l), respectively. The characterizations were evaluated by spectral analysis. In animal experiments, the enzymatic activities, lipid peroxide contents, and serum lipid parameters were measured, and histological observations of the liver, kidney, pancreas, and heart were conducted. The results demonstrated that treatment with En-, Ac-, and Al-MSP increased the organ enzymatic activities, decreased the organ lipid peroxide contents, mitigated the serum biochemistry values, and ameliorated the histopathology of diabetic mice, indicating that En-, Ac-, and Al-MSP could potentially be used as functional foods for the prevention of diabetes.