Foxa1 (formerly hepatic nuclear factor 3alpha) belongs to the family of Foxa genes that are expressed in early development and takes part in the differentiation of endoderm-derived organs and the regulation of glucose homeostasis. Foxa1-/- pups are growth retarded and hypoglycemic but glucose intolerant in response to an intraperitoneal glucose challenge. However, the mechanism of glucose intolerance in this model has not been investigated. Here, we show that Foxa1-/- islets exhibit decreased glucose-stimulated insulin release in islet perifusion experiments and have significantly reduced pancreatic insulin and glucagon content. Moreover, Foxa1-/- beta-cells exhibit attenuated calcium influx in response to glucose and glyburide, suggesting an insulin secretion defect either at the level or upstream of the ATP-sensitive K+ channel. Intracellular ATP levels after incubation with 10 mmol/l glucose were about 2.5 times lower in Foxa1-/- islets compared with controls. This diminished ATP synthesis could be explained by increased expression of the mitochondrial uncoupling protein uncoupling protein 2 (UCP2) in Foxa1-deficient islets, resulting in partially uncoupled mitochondria. Chromatin immunoprecipitation assays indicate that UCP2 is a direct transcriptional target of Foxa1 in vivo. Thus, we have identified a novel function for Foxa1 in the regulation of oxidative phosphorylation in pancreatic beta-cells.