Intraepithelial lymphocytes (IELs) reside in the epithelial layer to protect against foreign pathogens and maintain epithelial barrier functions in the intestine. The interaction between IELs and epithelial cells is required for their functions; however, the underlying molecular machinery still remains to be elucidated. Here, we found that intestinal epithelium-specific deficiency in a clathrin adaptor protein (AP)-1B, which regulates the basolateral protein sorting, led to the massive reduction of IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins—E-cadherin, Btnl2, and Plexin B2—decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among the downregulated proteins, CD166 is included. CD166 interacted with CD6 on the surface of induced IELs. CD166 knockdown using shRNA in intestinal organoid culture significantly inhibited the recruitment of IELs to an epithelial layer. These data suggest that basolateral sorting by AP-1B is indispensable for maintaining IELs in the epithelial layer and survival of IELs.