Glycolipids had received increasing attention because of their uses in various industries like cosmetics, pharmaceuticals, food and machinery manufacture. Microalgae were competitive organisms to accumulate metabolic substance. However, using microalgae to produce glycolipid was rare at present. In this study, glycolipid content of Chlorella pyrenoidosa and Synechococcus sp. under different nitrate and hydrophosphate levels were investigated. The highest glycolipid contents of 24.61% for C. pyrenoidosa and 15.37% for Synechococcus sp. were obtained at nitrate absence, which were 17.19% for C. pyrenoidosa and 10.99% for Synechococcus sp. at 0.01 and 0 g L(-1) hydrophosphate, respectively. Glycolipid productivities of two microalgae could reach at more than 10.59 mg L(-1) d(-1). Nitrate absence induced at least 8.5% increase in MGDG, DGDG and SQDG, while hydrophosphate absence resulted in over 21.2% increase in DGDG and over 48.4% increase in SQDG and more than 22.2% decrease in MGDG in two microalgae. Simultaneous nitrate and hydrophosphate limitation could make further improvement of glycolipid accumulation, which was more than 25% for C. pyrenoidosa and 21% for Synechococcus sp. These results suggest that nitrogen and phosphorus limitation or starvation should be an efficient way to improve microalgal glycolipid accumulation.