Abstract Optimization of medium ingredients for the production of mycelial polysaccharides from Phellinus baumii Pilat (PBMP) in submerged culture, preliminary characterization and the evaluation of antioxidant activity in vivo of PBMP were carried out. An optimal medium for PBMP production was obtained through a 2 (7−3) fractional factorial design and a central composite design in response surface methodology as follows (g/l): glucose 35.36, yeast extract 2.88, peptone 2.73, MgSO 4 1.47, KH 2 PO 4 1, VB 1 0.0075 and diammonium oxalate monohydrate 0.3. The resulting PBMP was composed of d -glucose, d -galactose, l -fucose, d -mannose, and l -rhamnose in a molar ratio of 12.74:1.39:1.00:1.92:0.22. It was demonstrated that the administration of PBMP could increase the activities of antioxidant enzymes, decrease the levels of malondialodehyde, and enhance total antioxidant capabilities in livers and serums of d -galactose-induced mice. The results suggested that PBMP had potent antioxidant activity and could be explored as novel natural antioxidant.