The effects of initial xylose concentration and nutritional supplementation of brewer's spent grain hydrolysate on xylitol production by Candida guilliermondii were evaluated using experimental design methodology. The hydrolysate containing 55, 75 or 95g/l xylose, supplemented or not with nutrients (calcium chloride, ammonium sulfate and rice bran extract), was used as fermentation medium. The increase in xylitol yield and productivity was related to the increase of initial xylose concentration, but up to a certain limit, above of which the yeast performance was not improved. The hydrolysate supplementation with nutrients did not interfere with xylose-to-xylitol conversion. By using the statistic tool the best conditions for maximum xylitol production were found, which consisted in using the non-supplemented hydrolysate containing 70g/l initial xylose concentration. Under these conditions, a xylitol yield of 0.78g/g and productivity of 0.58g/(lh) were achieved.