Pancreatic beta-cells are poised to sense glucose and other nutrient secretagogues to regulate insulin exocytosis, thereby maintaining glucose homeostasis. This process requires translation of metabolic substrates into intracellular messengers recognized by the exocytotic machinery. Central to this metabolism-secretion coupling, mitochondria integrate and generate metabolic signals, thereby connecting glucose recognition to insulin exocytosis. In response to a glucose rise, nucleotides and metabolites are generated by mitochondria and participate, together with cytosolic calcium, to the stimulation of insulin release. This review describes the mitochondrion-dependent pathways of regulated insulin secretion. Mitochondrial defects, such as mutations and reactive oxygen species production, are discussed in the context of beta-cell failure that may participate to the etiology of diabetes.