Response surface methodology was employed to optimize the medium compositions for the production of mycelial polysaccharide from Pholiota dinghuensis Bi (PDP). As a result, the optimal medium for crude PDP production was determined as follows (g/L) glucose 36.0, corn flour 11.8, peptone 3.0, yeast extract 5.4, KH(2)PO(4) 1.0 and MgSO(4) 1.5. In a verification experiment, a yield of 756 ± 38 mg/L crude PDP was obtained. Furthermore, biochemical assay and histopathological analysis showed that crude PDP exerted significant hepatoprotective effect in a dose-dependent manner against carbon tetrachloride (CCl(4))-induced acute liver injury. Crude PDP prevented the increase of activities of serum alanine aminotransferase and aspartate aminotransferase, reduced the formation of malondialdehyde and enhanced the activities of superoxide dismutase and glutathione peroxidase in CCl(4)-induced hepatotoxicity in mice.