Gut hormones have many key roles in the control of metabolism, as they target diverse tissues involved in the control of intestinal function, insulin secretion, nutrient assimilation and food intake. Produced by scattered cells found along the length of the intestinal epithelium, gut hormones generate signals related to the rate of nutrient absorption, the composition of the luminal milieu and the integrity of the epithelial barrier. Gut hormones already form the basis for existing and developing therapeutics for type 2 diabetes mellitus and obesity, exemplified by the licensed glucagon-like peptide 1 (GLP1) mimetics and dipeptidyl peptidase inhibitors that enhance GLP1 receptor activation. Modulating the release of the endogenous stores of GLP1 and other gut hormones is thought to be a promising strategy to mimic bariatric surgery with its multifaceted beneficial effects on food intake, body weight and blood glucose levels. This Review focuses on the molecular mechanisms underlying the modulation of gut hormone release by food ingestion, obesity and the gut microbiota. Depending on the nature of the stimulus, release of gut hormones involves recruitment of a variety of signalling pathways, including G protein-coupled receptors, nutrient transporters and ion channels, which are targets for future therapeutics for diabetes mellitus and obesity.