Effect of NaCl-induced osmotic stress on lipid production was investigated in batch culture of Chlorella vulgaris. Based on the facts that NaCl stress improved lipid production but inhibited cells growth at the same times, the novel strategies of multiple osmotic stresses with different NaCl additions (2 g/L at 80 h, 4 g/L at 100 h, and 6 g/L at 120 h) were adopted for lipid overproduction. Results show that after 180 h cultivation, lipid yield reached 3.16 g/L and intracellular lipid content was 58.6%, increased by 21.1 and 22.9%, respectively, compared to the control. Further applying the strategies to 5 L fermentor, lipid yield of 3.81 g/L was achieved at 180 h, which was 30.1% higher than the control, suggesting application of osmotic stress to lipid overproduction as being feasible.Key words: NaCl-induced osmotic stress, heterotrophic cultivation, lipid, glucose, Chlorella vulgaris