In the present work, optimization of microwave-assisted extraction of polysaccharides from Sargassum thunbergii was investigated. The physicochemical properties, structural characteristics, and in vitro antioxidant and hypoglycemic activities were determined. The optimal extraction conditions with a yield of 2.84±0.09% for extraction of polysaccharides (STP-1) were extraction time 23min, microwave power 547W, extraction temperature 80°C, and the ratio of raw material to water 1:27g/mL. STP-1 contained 32.7% of total carbohydrate, 1.86% of protein, and 15.2% of sulfates. STP-1 had a major molecular weight of 190.4kDa, and comprised of arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid with molar percentages of 1.94, 30.7, 4.54, 23.2, 17.6, 8.11, and 13.9%, respectively. In addition, STP-1 showed strong antioxidant and α-glucosidase inhibitory activities, and could improve the glucose uptake in insulin-resistant HepG2 cells, suggesting that STP-1 can be exploited as a promising natural antioxidant and hypoglycemic agent.