Xylose reductase activity of Candida guilliermondii FTI 20037 was evaluated during xylitol production by fed-batch fermentation of sugarcane bagasse hydrolysate. A 2(4-1) fractional factorial design was used to select process variables. The xylose concentrations in the feeding solution (S-F) and in the fermentor (So), the pH, and the aeration rate were selected for optimization of this process, which will be undertaken in the near future. The best experimental result was achieved at S-F = 45 g/L, S-0 = 40 g/L, pH controlled at 6.0, and aeration rate of 1.2 vvm. Under these conditions, the xylose reductase activity was 0.81 U/mg of protein and xylitol production was 26.3 g/L, corresponding to a volumetric productivity of 0.55 g/(L(.)h) and a xylose xylitol yield factor of 0.68 g/g.