Response surface (RSM) methodology based on a three-level three-factor Box-Behnkendesign of experiment was used to optimize the exopolysaccharide content (EPC) and themycelium biomass in submerged cultivation by Clitocybe sp. AS 5.112. The criticalfactors selected for the investigation were cultivation temperature, time and volume ofmedium, based on the results of previous Plackett-Burman design. By analyzing theresponse surface plots, the optimum ranges of cultivation temperature, time and mediumvolume for obtaining over 1 253.00gmL-1 of EPC lie in 24.325.8℃, 9.710.2d and 76.090.0mL, respectively. While for obtaining over 8.32 mg mL-1 of dry cell weight (DCW), theabove variables would be in the range of 23.824.8℃, 9.610.3d and 71.098.0mL,respectively. By solving the inverse matrix from the quadratic regression equations, theoptimal conditions to gain 1 265.45gmL-1 of EPC were 25.0℃, 9.9d and 83.4mL, to gain8.50mg mL-1 of DCW were 24.4℃, 9.9d and 87.1mL. In order to obtain the maximum yield ofEPC and DCW at the same time, the above conditions would be 24.5℃, 9.9d and 84.7mL,respectively, in this situation, the maximum predicted EPC and DCW were 1 261.60gmL-1and 8.47mgmL-1, respectively. The experimental data under various conditions have validatedthe theoretical values. Response surface (RSM) methodology based on a three-level three-factor Box-Behnkendesign of experiment was used to optimize the exopolysaccharide content (EPC) and themycelium biomass in submerged cultivation by Clitocybe sp. AS 5.112. The critical factors selected for the investigation were cultivation temperature, time and volume of medium, based on the results of previous Plackett-Burman design. By analyzing the response surface plots, the optimum ranges of cultivation temperature, time and medium volume for obtaining over 1 253.00 g mL-1 of EPC lie in 24.325.8 ° C, 9.710.2d and 76.090.0mL, respectively. While for obtaining over 8.32 mg mL-1 of dry cell weight (DCW), theabove variables would be in the range of 23.824.8 ° C, 9.610.3d and 71.098.0 mL, respectively. By solving the inverse matrix from the quadratic regression equations, the optimal conditions to gain 1 265.45 g mL-1 of EPC were 25.0 ° C., 9.9 d and 83.4 mL, to gain 8.50 mg mL-1 of DCW were 24.4 ° C., 9.9 d and 87.1 mL. In order to obtain the maximum yield of EPC and DCW at the same time, the above conditions would be 24.5 ° C., 9.9 d and 84.7 mL, respectively, in this situation, the maximum predicted EPC and DCW were 1 261.60 g mL-1 and 8.47 mg mL -1, respectively. experimental data under various conditions have validated the theoretical values.