AbstractThe extraction parameters of intracellular polysaccharide (IPS) from Hypsizigus marmoreus SK-01 mycelia were optimized, and the antioxidant activities of IPS were investigated. The optimum conditions of IPS extraction were predicted to be: an ultrasonic treatment time of 618.07 s, an extraction temperature of 84.53 °C, a pH of 7.57, and an IPS yield of ca. 6.84%. The in vitro inhibition effects of IPS on hydroxyl, superoxide anion, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were 52.63 ± 3.19%, 68.21 ± 5.09%, and 63.43 ± 5.27%, respectively, and the EC50 values of IPS were 0.85 ± 0.05 g/L, 0.47 ± 0.03 g/L, and 0.62 ± 0.04 g/L, respectively. The activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in mice blood were 2.52 ± 0.02 U/mL, 453.19 ± 38.43 U/mL, and 118.59 ± 9.64 U/mL, respectively. The results provide a reference for large-scale extraction of IPS by H. marmoreus SK-01 in industrial fermentation, suggesting that IPS can be used as a potential antioxidant that enhances adaptive immune responses.