Sorbitol is a sugar polyol claimed to have health-promoting properties. D-sorbitol-6-phosphate dehydrogeanse (StolPDh) is required for sorbose and sorbitol metabolism in Lactobacillus casei. StolPDh catalyzes the oxidation of Sorbitol-6-phosphate and also the reverse reaction, or reather, the reduction of fructose-6-phosphate with NAD+ regeneration. In order to test this function in vivo, a new food grade recombinant strain of L. casei was constructed by the integration of a StolPDh-encoding gene (gutF) in the chromosomal lactose operon. Lactose induction of gutF expression was achieved and sorbitol production could be detected by 13C-nuclear magnetic resonance analys, upon addiction of glucose to resting cells. Additional inactivationn of L-lactate dehydrogenase led to higher sorbitol production. These outcomes indicates that the accumulation of the glycolytic precursor, fructose-6-phosphate and an high NAD+/NADH ratio were the limiting factors for sorbitol accumulation.