Response surface methodology (RSM) was used for statistical optimization of fermentation medium that influenced the yield of endo-polysaccharide from cultivated mycelia of Cordyceps militaris. First, the Plackett-Burman design was used to evaluate the effects of ten variables including glucose, maltose, peptone, yeast extract, KH2PO4, MgSO4, CaCl2, VB1, inoculum density and medium capacity. Among these variables, glucose, peptone and yeast extract were identified to have the significant effects. Subsequently, response surface methodology based on a five-level three-factor central composite design was employed to determine the maximum dry weight (DW) of mycelial biomass at optimum concentration of glucose, peptone and yeast extract. The mycelia growth was found to correlate to the three parameters that could be represented by second-order polynomial models. The optimal values of the three parameters were determined as 4.62% glucose, 3.36% peptone and 0.43% yeast extract. The prediction DW was 23.727g/L. The actual experimental results were in agreement with the prediction.