Ypsizigus marmoreus is a newly cultivated medicinal and edible mushroom with higher antitumor activity. In this paper, a uniform design was used to optimize the medium formula with four factors each at six levels, and a regression model was constructed to predict the antitumor exopolysaccharide production. The optimal concentrations for the enhanced exopolysaccharide production were determined as 30% (w/v) glucose, 8.4% (w/v) soybean peptone, 3% (w/v) KH2PO4 and 30% (w/v) sweat potato flour. Subsequent experiment confirmed the validity of the model. Under the optimal condition, exopolysaccharide yield reached 3.39g/L in shake-flask culture, which was 3.53 fold higher than that obtained in the basic medium. The results highlight the future application of optimized medium for high yield production of antitumor exopolysaccharide by fermentation of Hypsizigus marmoreus.