Statistically based experimental designs were applied to the optimization of medium composition for exo-polysaccharide production by Cordyceps militaris NG3 in shake-flask cultures. First, the Plackett-Burman design was used to search for the main factors on mycelia and exo-polysaccharide production. Among these variables, sucrose and corn steep powder were found to be two significant factors and had positive effects on mycelial yield (with confidence level >80%) and exo-polysaccharide production (with confidence level >90%). Subsequently, to study the mutual interactions between variables, the effects of the two main factors on exo-polysaccharide production were further investigated using a central composite design. The optimal composition was found to be 1.03 g/l corn steep powder, 2.95 g/l sucrose, 0.1 g/l K(2)HPO(4), 0.5 g/l MgSO(4) x 5H(2)O and 0.1 g/l KNO(3) for the enhanced production of the exo-polysaccharide, which was 2.604 g/l in shake-flask cultures. Under optimal culture conditions, the maximum exo-polysaccharide concentration in a 5 l stirred-tank bioreactor was 3.8 g/l.