A potential continuous nitrogen-limitation (CNL) strategy was proposed to enhance lipid accumulation by oleaginous Chlorella vulgaris in this study. The highest biomass (4.61 g/L) with CNL was nearly close to the maximum biomass (4.88 g/L) achieved by the traditional batch nitrogen-starvation strategy (BNS) under laboratory condition. CNL was further found to diminish the photosynthetic activity and trigger the degradation of nitrogenous compounds (e.g. protein, chlorophyll, DNA) consequently resulted in enhanced lipid accumulation. The maximal lipid productivity of 305.71 mg/L/d was accomplished by CNL, which presented 1.35-fold more than that of BNS. Furthermore, the identified lipid accumulation-related metabolic checkpoints (MDH and GPDH) provide the possibilities to develop high-lipid engineering microalgae. The aforementioned results imply that CNL nitrogen-deficiency strategy for lipid production on large-scale deserves further exploration. (C) 2015 Elsevier Ltd. All rights reserved.