The hemicellulosic fraction of brewer's spent grain (BSG) was hydrolysed with diluted acid under different conditions of liquid/solid ratio (8–12 g g−1), sulfuric acid concentration (100–140 mg g−1 dry matter) and reaction time (17–37 min) in order to produce a liquor with a large amount of xylose and good fermentability to produce xylitol. Results showed that all the evaluated reaction conditions were able to hydrolyse xylan and arabinan with efficiencies higher than 85.8 and 95.7% respectively, and even under the mildest reaction condition a considerable amount (92.7%) of the hemicellulosic fraction could be extracted. The hydrolysates presented different fermentabilities when used as fermentation media for xylitol production by Candida guilliermondii yeast, owing to the differences in their composition. Based on statistical analysis, the best condition for BSG acid hydrolysis was the use of a liquid/solid ratio of 8 g g−1, 100 mg H2SO4 g−1 dry matter and a reaction time of 17 min. Under this condition a high extraction efficiency of hemicellulosic sugars (92.7%) and good fermentation results (YP/S = 0.70 g g−1 and QP = 0.45 g dm−3 h−1) were attained.