Objective The extraction of the hemicellulose fraction of sugarcane bagasse (SCB) by acid hydrolysis was evaluated in an autoclave and a Parr reactor aiming the application of the hydrolysate as a carbon source for lipid production by Lipomyces starkeyi. Results The hydrolysis that resulted in the highest sugar concentration was obtained by treatment in the Parr reactor (HHR) at 1.5% (m/v) H2SO4 and 120 degrees C for 20 min, reaching a hemicellulose conversion of approximately 82%. The adaptation of the yeast to the hydrolysate provided good fermentability and no lag phase. The fermentation of hemicellulose-derived sugars (HHR) by L. starkeyi resulted in a 27.8% (w/w) lipid content and Y-P/S of 0.16 g/l.h. Increasing the inoculum size increased the lipid content by approximately 61%, reaching 44.8% (w/w). Conclusion The hemicellulose hydrolysate from SCB is a potential substrate for L. starkeyi to produce lipids for biodiesel synthesis based on the biorefinery concept.