Intestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn’s disease (CD). Mechanisms controlling colonocyte differentiation and barrier defects are understudied in CD. We recently reported increased expression of microRNA-31-5p (miR-31-5p) in colonic IECs of CD patients compared to non-IBD (NIBD) controls. In this study we identify and characterize the miR-31-5p target gene Activin Receptor-Like Kinase 1 (ALK1) as a key regulator of colonocyte maturation and IEC barrier integrity, specifically the functional impact of aberrant ALK1 signaling on colonocyte differentiation, barrier integrity, and clinical disease outcomes in CD.