Background: Diabetes is related with oxidative stress caused by free radicals and glycation of the proteins. Objectives: This study aimed to isolate a polysaccharide (PLY) from Cucurbita argyrosperma seeds and determine their antioxidant and antiglycation effects. Materials and Methods: The PLY fraction was isolated by hot water extraction and purified by ion-exchange chromatography (A103S). La antioxidant capacities were evaluated by using lipid peroxidation, superoxide dismutase, glutathione peroxidase (GSH-Px), catalase, and malondialdehyde activities, and intracellular reactive oxygen species (ROS) with stimulation H2O2 in pancreatic β INS-1 cells. In addition, scavenging activities of PLY were estimated in 2,2 diphenyl-1-picrylhydrazyl radical, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, β-carotene-linoleic acid, chelating activity, ferric reducing power, hydroxyl radical, nitric oxide radical, and superoxide anion radical assays. Therefore, the protective effect of PLY on reducing sugar-induced bovine serum albumin glycation was examined. Results: PLY consisted of mannose, galactose, and glucose in the molar ratio of 1.12:6.12:5.60 with an average molecular weight of 102326 Da. PLY had a pronounced radical scavenging potential as well as total antioxidant effect and prevented the cells ROS generation and increased the level of antioxidant enzymes. PLY inhibited the formation of carboxymethyllysine and fructosamine, prevented protein oxidation increasing the level of thiol, and decreasing protein carbonyl level, interacts with reactive dicarbonyl compounds (methylglyoxal), and decreased amyloid cross β-structure level. Conclusion: These results indicate that PLY enhanced antioxidant capacity and prevented oxidative stress and AGEs progression in its different stages. consequently, had promising effects on diabetes and associated disorders.