Intestine‐on‐chip has been developed to study intestinal physiology and pathophysiology as well as intestinal transport absorption and toxicity studies in a controlled and human similar environment. Here we report that dynamic culture of an Intestine‐On‐Chip enhances ECM remodeling of the stroma, the basement membrane production and speeds up epithelial differentiation. We developed a 3D human intestinal stromal equivalent (3D‐ISE) composed of human intestinal subepithelial myofibroblasts (ISEMFs) embedded in their own extracellular matrix. Then, we cultured human colon carcinoma‐derived cells (Caco‐2) in both static and dynamic conditions in the opportunely designed microfluidic system until the formation of a well‐oriented epithelium. This low cost and handy microfluidic device allows to qualitatively and quantitatively detecting epithelial polarization, mucus production as well as the monitoring …