The winged-helix transcription factor Foxa2 regulates Pdx1 gene expression and fetal endocrine pancreas development. We show here by inducible gene ablation that Foxa2 inactivation in mature β cells induces hyperinsulinemic hypoglycemia in Foxa2^loxP/loxP,Pdx1-CreERT2 adult mice. Mutant β cells exhibited a markedly increased pool of docked insulin granules, some of which were engaged in sequential or compound exocytosis, consistent with increased first-phase glucose-stimulated insulin secretion. Expression of multiple genes involved in vesicular trafficking, membrane targeting, and fuel-secretion pathways is dependent on Foxa2. In addition, impaired cytosolic Ca²⁺ oscillations and elevated intracellular cyclic AMP production accompanied this secretory defect and were likely contributors to the sensitization of the exocytotic machinery. Thus, in the absence of Foxa2, alterations in intracellular second-messenger signaling redistribute the insulin granules into the readily releasable pool. We conclude that Foxa2 is required for both fetal pancreas development and the function of mature β cells.