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Serotonin (5-hydroxytryptamine, 5-HT) is an important multifunctional bioamine, with roles in a range of physiological pathways. Almost all of the 5-HT in our body, and all of the circulating 5-HT, is synthesized and secreted by specialized enteroendocrine cells within the gastrointestinal (GI) mucosa, called enterochromaffin (EC) cells. EC cell–derived 5-HT has a wide array of actions within the gut, including modulating GI motility. Recent evidence demonstrates that peripheral 5-HT also plays a key role outside of the gut, affecting platelet clotting (see chapter by Schenwalder et al.), energy metabolism, and glucose homeostasis (see chapter by Xu et al.). Accordingly, factors that directly influence EC cell 5-HT synthesis and secretion, and alter peripheral 5-HT, have implications in a number of GI and metabolic disorders such as type 2 diabetes and obesity where energy homeostasis is significantly perturbed. This review discusses the role of EC cells as important sensory cells within the intestinal tract and how nutrient sources derived from diet and gut microbiota regulate gut 5-HT, and it highlights the newfound importance of EC cells in human diseases associated with metabolic dysfunction.