Xylitol is a sweetener with important technological properties like anticariogenicity, low caloric value, and negative dissolution heat. Because it can be used successfully in food formulations and pharmaceutical industries, its production is in great demand.Xylitol can be obtained by microbiological process, since many yeasts and filamentous fungi synthesize the xylose reductase enzyme, which catalyses the xylose reduction into xylitol as the first step in the xylose metabolism. The xylitol production by biotechnological means has several economic advantages in comparison with the conventional process based on the chemical reduction of xylose. The efficiency and the productivity of this fermentation chiefly depends upon the microorganism and the process conditions employed. In this mini-review, the most significant upstream parameters on xylitol production by biotechnological process are described.