Consumption of a high-protein diet increases protein entry into the colon. Colonic microbiota can ferment proteins, which results in the production of protein fermentation end-products, like polyamines. This review describes the effects of polyamines on biochemical, cellular and physiological processes, with a focus on the colon. Polyamines (mainly spermine, spermidine, putrescine and cadaverine) are involved in the regulation of protein translation and gene transcription. In this, the spermidine-derived hypusination modification of EIF5A plays an important role. In addition, polyamines regulate metabolic functions. Through hypusination of EIF5A, polyamines also regulate translation of mitochondrial proteins, thereby increasing their expression. They can also induce mitophagy through various pathways, which helps to remove damaged organelles and improves cell survival. In addition, polyamines increase mitochondrial substrate oxidation by increasing mitochondrial Ca2+-levels. Putrescine can even serve as an energy source for enterocytes in the small intestine. By regulating the formation of the mitochondrial permeability transition pore, polyamines help maintain mitochondrial membrane integrity. However, their catabolism may also reduce metabolic functions by depleting intracellular acetyl-CoA levels, or through production of toxic by-products. Lastly, polyamines support gut physiology, by supporting barrier function, inducing gut maturation and increasing longevity. Polyamines thus play many roles, and their impact is strongly tissue-and dose-dependent. However, whether diet-derived increases in colonic luminal polyamine levels also impact intestinal physiology has not been resolved yet.