A new approach to the biotechnological production of xylitol from sugarcane bagasse using Candida guilliermondii cells immobilized on porous glass in a fluidized bed reactor (FBR) is presented. The influence of aeration rate (AR) and carrier concentration (Cs) on the production process was evaluated. Seven batch fermentation runs were conducted according to a 22 factorial design. The results obtained showed that Cs had a negative influence on xylitol yield (Yp/s) and volumetric productivity (Qp), whereas AR had a positive influence on Qp and a negative influence on Yp/s. With an AR=0.093 min−1 and Cs=62.5 g/l, the Yp/s value was low (0.25 g/g), but the Qp was highest (0.44 g/l h), probably because cell metabolism was faster when more oxygen was available to the yeast. When the highest levels of the AR and Cs were used, the xylitol yield and concentration were the lowest and the cell concentration was the highest, suggesting that cell metabolism was directed preferentially towards biomass production instead of xylitol accumulation.