DNA-binding transcription factors bind to promoters that carry their binding sites. Transcription factors therefore function as nodes in gene regulatory networks. In the present work we used a bioinformatic approach to search for transcription factors that might function as nodes in gene regulatory networks during the differentiation of the small intestinal epithelial cell. In addition we have searched for connections between transcription factors and the villus metabolome. Transcriptome data were generated from mouse small intestinal villus, crypt, and fetal intestinal epithelial cells. Metabolome data were generated from crypt and villus cells. Our results show that genes that are upregulated during fetal to adult and crypt to villus differentiation have an overrepresentation of potential hepatocyte nuclear factor (HNF)-4 binding sites in their promoters. Moreover, metabolome analyses by magic angle spinning (1)H nuclear magnetic resonance spectroscopy showed that the villus epithelial cells contain higher concentrations of lipid carbon chains than the crypt cells. These findings suggest a model where the HNF-4 transcription factor influences the villus metabolome by regulating genes that are involved in lipid metabolism. Our approach also identifies transcription factors of importance for crypt functions such as DNA replication (E2F) and stem cell maintenance (c-Myc).