An 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 extracellular matrix (ECM) remodeling of the stroma, basement membrane production and speeds up epithelial differentiation. We developed a three-dimensional human intestinal stromal equivalent composed of human intestinal subepithelial myofibroblasts embedded in their own ECM. Then, we cultured human colon carcinoma-derived cells 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 detect epithelial polarization and mucus production as well as monitor barrier function and ECM remodeling after nutraceutical treatment.