Sugarcane bagasse hemicellulosic hydrolysate (SCBH) is a low-cost substrate for single-cell oil (SCO) production. However, this hydrolysate has an undesirable low carbon/nitrogen (C/N) ratio and contains inhibitors. Yeast adaptation is a simple strategy to overcome the presence of inhibitors, while the concentration of the hydrolysate and glycerol supplementation are alternatives to solve the low C/N limitation. This work aimed to investigate the adaptation of the yeast Rhodosporidium toruloides in undetoxified SCBH and its use in SCO production. The adapted strain produced more lipids than the parental strain: the concentration of SCBH or the addition of glycerol increased the lipid content, concentration, and productivity to at least 108%, 175%, and 118%, respectively. Lipid production in SCBH was done without salts or nitrogen supplementation. Transcriptomic study showed that hydrolysate-tolerance- and lipid accumulation-related genes were strongly induced. These genes can be targets for metabolic engineering. (C) 2017 Elsevier Ltd. All rights reserved.