Abstract: Mushrooms have become attractive as a functional food and as a source for the development of drugs and nutraceuticals. Agaricus blazei is considered one of the best among them. There are several published works on the fruiting body of this mushroom, showing high antitumor activity. The mycelium polysaccharide (EPS) of this mushroom also demonstrated a strong antitumor action. However, there is a little information available in the literature about optimization of culture conditions for production of EPS by liquid submerged culture. A mathematical model was developed to show the effect of each medium composition and their interactions on the production of mycelial biomass and exo-polysaccharide. Study on supplementation of carbon source to the medium revealed that starch was the most effective for EPS production. Yeast extract was the best for EPS among the inorganic and organic nitrogen sources tested. The Box-Behnken model estimated that a maximal yield of mycelial biomass 9.08 g L-1 could be obtained when initial pH, temperature, starch and yeast extract concentrations were set at 5.64, 24.60°C, 11.55 and 4.63 g L-1, respectively while a maximal EPS yield of 1.268 g L-1 could be achieved when pH, temperature, starch and yeast extract concentrations were set at 6.87, 20°C, 20 and 0.84 g L-1, respectively. These predicted values also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yields of mycelial biomass and EPS.