Objective To investigate the optimal fermentation conditions for the production of antitumor active substances by marine bacteria HGX5 and preliminary study on their physicochemical properties.Methods Six factors including glucose,peptone,yeast extract,culture temperature,medium pH and agitation were investigated by Plackett-Burman Design, and the important factors including culture temperature,glucose and peptone were optimized by using response surface methodology(RSM).Results The optimal fermentation conditions were culture temperature 35℃,glucose 2.0g?L~(-1) and peptone 9.3g?L~(-1).The antitumor active substances showed better heat and pH stabilities via physicochemical experiments. Conclusion The adopted approach based on Plackett-Burman design and RSM was fit for the fermentation of this strain.