Because many peptide and peptide-mimetic drugs are substrates of peptide transporter 1, it is important to evaluate the peptide transporter 1-mediated intestinal absorption of drug candidates in the early phase of drug development. Although intestinal cell lines treated with inhibitors of peptide transporter 1 are widely used to examine whether drug candidates are substrates for peptide transporter 1, these inhibitors are not sufficiently specific for peptide transporter 1. In this study, to generate a more precise evaluation model, we established peptide transporter 1-knockout induced pluripotent stem cells (iPSCs) by using a CRISPR-Cas9 system and differentiated the cells into intestinal epithelial-like cells. The permeability value and uptake capacity of glycylsarcosine (substrate of peptide transporter 1) in peptide transporter 1-knockout intestinal epithelial-like cells were significantly lower than those in wild-type intestinal epithelial-like cells, suggesting that peptide transporter 1 was successfully depleted in the epithelial cells. Taken together, our model can be useful in the development of peptide and peptide-mimetic drugs.