Mitochondrial dysfunction is an important contributor to human pathology 1, 2, 3, 4 and it is estimated that mutations of mitochondrial DNA (mtDNA) cause approximately 0.5–1% of all types of diabetes mellitus 5, 6. We have generated a mouse model for mitochondrial diabetes by tissue-specific disruption of the nuclear gene encoding mitochondrial transcription factor A (Tfam, previously mtTFA; ref. 7) in pancreatic β-cells. This transcriptional activator is imported to mitochondria, where it is essential for mtDNA expression and maintenance 8, 9. The Tfam-mutant mice developed diabetes from the age of approximately 5 weeks and displayed severe mtDNA depletion, deficient oxidative phosphorylation and abnormal appearing mitochondria in islets at the ages of 7–9 weeks. We performed physiological studies of β-cell stimulus–secretion coupling in islets isolated from 7–9-week-old mutant mice and found reduced hyperpolarization of the mitochondrial membrane potential, impaired Ca 2+-signalling and lowered insulin release in response to glucose stimulation. We observed reduced β-cell mass in older mutants. Our findings identify two phases in the pathogenesis of mitochondrial diabetes; mutant β-cells initially display reduced stimulus–secretion coupling, later followed by β-cell loss. This animal model reproduces the β-cell pathology of human mitochondrial diabetes and provides genetic evidence for a critical role of the respiratory chain in insulin secretion.