The potential use of sugarcane bagasse hydrolysate (SBH) for microalgal oil in a heterotrophic mode and the oil accumulation mechanisms by SBH-induced Chlorella protothecoides cells were investigated in this study. Results demonstrated that SBH performed better than glucose for cell growth and lipid accumulation under the same reducing sugar concentration. The lipid productivity of 0.69 g/L/d was accomplished at 40 g/L of reducing sugar by batch culture. Under the fed-batch culture condition, the maximum biomass and lipid productivity were 24.01 g/L and 1.19 g/L/d, respectively. Metabolic pathway analysis results indicated that xylose and arabinose involved in pentose phosphate pathway might be predominant over sole glucose involved in glycolysis for lipid accumulation in cells. Three metabolic checkpoints in the proposed metabolic network, including xylulose kinase, acyl-CoA dehydrogenase, and dihydrolipoyl dehydrogenase reveal new possibilities in developing genetic and metabolic engineering microalgae for desirable lipid productivity. (C) 2015 Elsevier Ltd. All rights reserved.