The intestinal epithelium is a rapidly self-renewing tissue; the rapid turnover prevents the invasion of pathogens and harmful components from the intestinal lumen, preventing inflammation and infectious diseases. Intestinal epithelial barrier function depends on the epithelial cell proliferation and junctions, as well as the state of the immune system in the lamina propria. Polyamines, particularly putrescine, spermidine, and spermine, are essential for many cell functions and play a crucial role in mammalian cellular homeostasis, such as that of cell growth, proliferation, differentiation, and maintenance, through multiple biological processes, including translation, transcription, and autophagy. Although the vital role of polyamines in normal intestinal epithelial cell growth and barrier function has been known since the 1980s, recent studies have provided new insights into this topic at the molecular level, such as eukaryotic initiation factor-5A hypusination and autophagy, with rapid advances in polyamine biology in normal cells using biological technologies. This review summarizes recent advances in our understanding of the role of polyamines in regulating normal, non-cancerous, intestinal epithelial barrier function, with a particular focus on intestinal epithelial renewal, cell junctions, and immune cell differentiation in the lamina propria.