The neural cell adhesion molecule (NCAM) is required for cell type segregation during pancreatic islet organogenesis. We have investigated the functional consequences of ablating NCAM on pancreatic β-cell function. In vivo, NCAM^−/− mice exhibit impaired glucose tolerance and basal hyperinsulinemia. Insulin secretion from isolated NCAM^−/− islets is enhanced at glucose concentrations below 15 mM but inhibited at higher concentrations. Glucagon secretion from pancreatic α-cells evoked by low glucose was also severely impaired in NCAM^−/− islets. The diminution of insulin secretion is not attributable to defective glucose metabolism or glucose sensing (documented as glucose-induced changes in intracellular Ca²⁺ and K_ATP-channel activity). Resting K_ATP conductance was lower in NCAM^−/− β-cells than wild-type cells, and this difference was abolished when F-actin was disrupted by cytochalasin D (1 μM). In wild-type β-cells, the submembrane actin network disassembles within 10 min during glucose stimulation (30 mM), an effect not seen in NCAM^−/− β-cells. Cytochalasin D eliminated this difference and normalized insulin and glucagon secretion in NCAM^−/− islets. Capacitance measurements of exocytosis indicate that replenishment of the readily releasable granule pool is suppressed in NCAM^−/− α- and β-cells. Our data suggest that remodeling of the submembrane actin network is critical to normal glucose regulation of both insulin and glucagon secretion.