<p>Regulation of insulin secretion by pancreatic β-cells is central to maintaining glucose homeostasis and overall metabolic health. Glucose is recognized as the major driver of insulin secretion; however, here, we highlight established and emerging evidence of important contributions of other nutrients, including amino acids, fatty acids and alternative fuels, as well as non-nutrient factors, in modulating both basal and postprandial insulin release. We discuss molecular mechanisms underlying insulin release, including roles for reactive oxygen species, and the consequences of β-cell overwork. This Review also addresses the physiological and pathophysiological significance of insulin secretion dynamics, including changes in β-cell mass and function across the lifespan, circadian and pulsatile patterns of secretion, and adaptive responses to metabolic stress. We reflect on how genetic predisposition, environmental exposures, pharmaceuticals, food additives and lifestyle factors can influence insulin secretion and contribute to hyperinsulinaemia, insulin resistance and metabolic disease. We identify knowledge gaps that would benefit from additional study, especially in human models. By integrating evidence from both human in vitro studies and preclinical models, we summarize the multifactorial regulation of insulin secretion and underscore its relevance for developing strategies to prevent and manage diabetes mellitus and related metabolic disorders.</p>