Pancreatic beta-cells are able to respond to nutrients, principally glucose, as the primary stimulus for insulin exocytosis. This unique feature requires translation of metabolic substrates into intracellular messengers recognized by the exocytotic machinery. Central to this signal transduction mechanism, mitochondria integrate and generate metabolic signals, thereby coupling glucose recognition with insulin secretion. In response to a glucose rise, nucleotides and metabolites are generated by mitochondria and participate, together with cytosolic Ca2+, in the stimulation of insulin exocytosis. Mitochondrial defects, such as mutations and ROS (reactive oxygen species) production, might be associated with beta-cell failure in the course of diabetes. mtDNA (mitochondrial DNA) mutation A3243G is associated with MIDD (mitochondrial inherited diabetes and deafness). A common hypothesis to explain the link between the genotype and the phenotype is that the mutation might impair mitochondrial metabolism expressly required for beta-cell functions, although this assumption lacks direct demonstration. mtDNA-deficient cellular models are glucose-unresponsive and are defective in mitochondrial function. Recently, we used clonal cytosolic hybrid cells (namely cybrids) harbouring mitochondria derived from MIDD patients. Compared with control mtDNA from the same patient, the A3243G mutation markedly modified metabolic pathways. Moreover, cybrid cells carrying patient-derived mutant mtDNA exhibited deranged cell Ca2+ handling and elevated ROS under metabolic stress. In animal models, transgenic mice lacking expression of the mitochondrial genome specifically in beta-cells are diabetic and their islets are incable of releasing insulin in response to glucose. These various models demonstrate the fragility of nutrient-stimulated insulin secretion, caused primarily by defective mitochondrial function.