Using Plackett–Burman design, steepest ascent method, and Box–Behnken design, we carried out sequential statistical optimization of the Phellinus nigricans culture medium for maximizing the production of exopolysaccharides. The concentrations of the constituents of culture medium as optimized by this method were: 51.67g/L glucose, 6.96g/L peptone, 1.0g/L yeast extract, 5mg/L thiamine, 1.0g/L KH2PO4, and 0.5g/L MgSO4. The yield of exopolysaccharides obtained using the optimized culture medium was 1.89±0.13g/L—2.3-fold higher than that obtained when unoptimized culture medium was used. The exopolysaccharide of P. nigricans (PNEP) is comprised by glucose and mannose with the molar ratio 1:2.63. PNEP thus produced scavenged superoxide anion radical, 1,1-diphenyl-2-picrylhydrazyl radical, and hydroxyl radical; showed antioxidant properties; and had reducing power.