- BACKGROUND AND AIMS: Although Hnf1alpha is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions. The aim of this study was to assess the consequences of abrogating Hnf1alpha on the maintenance of adult small intestinal epithelial functions. METHODOLOGY/PRINCIPAL FINDINGS: An Hnf1alpha knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals. Changes in global gene expression were also analyzed. Mice lacking Hnf1alpha displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life. This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1alpha mutant mice. The intestinal epithelium of Hnf1alpha null mice displayed a reduction of the enteroendocrine cell population. An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway. CONCLUSIONS/SIGNIFICANCE: Together, these results unravel a functional role for Hnf1alpha in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages.