Polyamines regulate a variety of physiological functions and are involved in pathogenesis of diverse human diseases. The epithelium of the mammalian gut mucosa is a rapidly self-renewing tissue in the body, and its homeostasis is preserved through well-controlled mechanisms. Here, we highlight the roles of cellular polyamines in maintaining the integrity of the gut epithelium, focusing on the emerging evidence of polyamines in the regulation of gut epithelial renewal and barrier function. Gut mucosal growth depends on the available supply of polyamines to the dividing cells in the crypts, and polyamines are also essential for normal gut epithelial barrier function. Polyamines modulate expression of various genes encoding growth-associated proteins and intercellular junctions via distinct mechanisms involving RNA-binding proteins and noncoding RNAs. With the rapid advance of polyamine biology, polyamine metabolism and transport are promising therapeutic targets in our efforts to protect the gut epithelium and barrier function in patients with critical illnesses.