Response surface methodology (RSM) was employed to optimize culture medium for lipid production of Saccharomyces cerevisiae. Firstly, according to the results of mono-factors experiment, a Plackett-Burman design was used to investigate the effects of different factors in the ferment medium, three statistically significant factors are:citric acid, CaCl2, initial pH value, and then steepest ascent procedures was employed to define optimal response region for these three factors. Finally, Box-Behnken was employed to design. The results of experiments was analyzed by RSM with Minitab15.0 soft. The optimal lipid production medium is: glucose 15%, peptone 0.2%, yeast extraction 0.4%, citric acid 0.471%, MgSO4?7H2O 0.1%, ZnSO4?7H2O 0.2%, CaCl2 0.025%, FeSO4?7H2O 0.005%, initial pH value 6.74, 180 r/min, incubme in 30?C for 96 h. Lipid production (dry weight) after optimization is increased from 4.76% to 14.55%. The yield of lipid is increased by about 3 times.