Abstract The intestinal barrier is mainly formed by a monolayer of epithelial cells, which forms a physical barrier to protect the gut tissues from external insults and provides a microenvironment for commensal bacteria to colonize while ensuring immune tolerance. Moreover, various immune cells are known to significantly contribute to intestinal barrier function by either directly interacting with epithelial cells or by producing immune mediators. Fulfilling this function of the gut barrier for mucosal homeostasis requires not only the intrinsic regulation of intestinal epithelial cells (IECs) but also constant communication with immune cells and gut microbes. The reciprocal interactions between IECs and immune cells modulate mucosal barrier integrity. Dysregulation of barrier function could lead to dysbiosis, inflammation, and tumorigenesis. In this overview, we provide an update on the characteristics and functions of IECs, and how they integrate their functions with tissue immune cells and gut microbiota to establish gut homeostasis.