Human induced pluripotent stem (iPS) cells were differentiated into the endoderm using activin A and were then treated with fibroblast growth factor 2 (FGF2) for differentiation into intestinal stem cell-like cells. These immature cells were then differentiated into enterocyte-like cells using epidermal growth factor (EGF) in 2% fetal bovine serum (FBS). At the early stage of differentiation, mRNA expression of caudal type homeobox 2 (CDX2), a major transcription factor related to intestinal development and differentiation, and leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, was markedly increased by treatment with FGF2. When cells were cultured in medium containing EGF and a low concentration of FBS, mRNAs of specific markers of intestinal epithelial cells, including sucrase-isomaltase, the intestinal oligopeptide transporter SLC15A1/peptide transporter 1 (PEPT1), and the major metabolizing enzyme CYP3A4, were expressed. In addition, sucrase-isomaltase protein expression and uptake of β-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA), a fluorescence-labeled substrate of the oligopeptide transporter, were detected. These results demonstrate a simple and direct method for differentiating human iPS cells into functional enterocyte-like cells.