This work for the first time investigated lipid accumulation by a two-stage regime namely heterotrophic-Cu(II) stressed (HCuS) and underlying molecular basis of lipid biosynthesis in Chlorella protothecoides cells. The results showed that the optimized biomass and lipid yield were achieved by 6.47 g/L and 5.78 g/L with this strategy. The fatty acids compositions (almost 100% of them are C15 to C20) are ideal for preparing high quality biodiesel. Further, 30 differentially expressed proteins response to HCuS were involved in carbohydrate metabolism, carbon fixation, TCA cycle, lipid metabolism, protein biosynthesis, transportation and regulation, ATP and RNA biosynthesis, nucleotide metabolism, ROS scavenging. Especially, glycolysis pathway might be the important contributor for lipid accumulation. In future, further functional analysis of these altered proteins would help to reveal more concerning lipid biosynthesis pathway. (C) 2013 Elsevier Ltd. All rights reserved.