In the endocrine fraction of the pancreas, the task of the beta-cell is to continuously and perfectly adjust insulin secretion to fluctuating blood glucose levels, thereby maintaining glycemia and nutrient homeostasis. This glucose sensing coupled to insulin exocytosis depends on transduction of metabolic signals into intracellular messengers recognized by the exocytotic machinery. Central to this metabolism-secretion coupling, mitochondrial signal transduction refers to both integration and generation of metabolic signals, connecting glucose sensing to insulin exocytosis. In response to a glucose rise, nucleotides and metabolites are generated by mitochondria and participate, together with cytosolic calcium, in the stimulation of insulin release. This review describes the role of mitochondria in metabolic signal transduction regulating insulin secretion.