The sorbitol is a naturally occurring polyol, is widely used in the food industry as a sweetener in low calorie foods, humectant and texturizing agent. This sugar alcohol is used by some species of Lactobacillus and as carbon source by human intestinal bífidobacterias. For this reason, some authors have concluded that sorbitol could be a prebiotic. However, there are few experimental data concerning its potential as prebiotic. In this work, the in vivo effect of sorbitol has been analysed on cecal, colonic and fecal microbiota, using rats as an animal model, and its effect was compared to that of a well known prebiotic such as fructooligosaccharides (FOS).Intestinal microbiota was studied by classic cultural methods and updated molecular techniques, such as PCR followed by denaturing gradient gel electrophoresis (DGGE) and quantitatively by real-time PCR (qPCR). Resultsshowed that the consumption of sorbitol modified intestinal microbiota in a similar way to FOS, both qualitatively and quantitatively. Sorbitol increased and favored, respectively, the permanence of Lactobacillus reuteri and Lactobacillus sp. AD 102 in the gastrointestinal tract of the rats. Analysis of organic acid concentrations in the intestinal content showed that sorbitol intake significantly increased colonic and cecal butyrate levels. Therefore in our animal model, sorbitol has the ability of modifying the gut microbiota activity, possibly contributing to maintain a healthy colonic mucosa.Five Lactobacillus species have been isolated from the intestinal content of rats.