Hepatocyte nuclear factor 4 (HNF4) is a regulator of hepatocyte and pancreatic transcription. Hnf4 deletion in the mouse is embryonically lethal with severe defects in visceral endoderm formation. It has been concluded in the past that the role of Hnf4 in the developing colon was much less important than in the liver. However, the precise role of Hnf4 in the homeostasis of the small intestinal epithelium remains unclear. Our aim was to evaluate the potential of Hnf4 to support an intestinal epithelial phenotype. First, Hnf4 potential to dictate this phenotype was assessed in non-intestinal cell lines in vitro. Forced expression of Hnf4 in fibroblasts showed an induction of features normally restricted to epithelial cells. Combinatory expression of Hnf4 with specific transcriptional regulators of the intestine resulted in the induction of intestinal epithelial genes in this context. Second, the importance of Hnf4 in maintaining the homeostasis of the intestinal epithelium was investigated in mice. Mice conditionally deficient for intestinal Hnf4 developed normally throughout adulthood with an epithelium displaying normal morphological and functional structures with minor alterations. Subtle but statistical differences were observed at the proliferation and the cytodifferentiation levels. Hnf4 mutant mice displayed an increase in the number of goblet and enteroendocrine cells as compared to controls. Given the fundamental role of this transcription factor in other tissues, these findings dispute the crucial role for this regulator in the maintenance of intestinal epithelial cell function at a period of time that follows cytodifferentiation but may suggest a functional role in instructing cells to become specific to the intestinal epithelium.