Cellular redox state is an important metabolic variable, influencing many aspects of cell function like growth, apoptosis, and reductive biosynthesis. In this report, we identify NADPH as a candidate signaling molecule for exocytosis in neuroendocrine cells. In pancreatic beta-cells, glucose acutely raised the NADPH-to-NADP+ ratio and stimulated insulin release in parallel. Furthermore, intracellular addition of NADPH directly stimulated exocytosis of insulin granules. Effects of NADPH on exocytosis are proposed to be mediated by the redox proteins glutaredoxin (GRX) and thioredoxin (TRX) on the basis of the following evidence: 1) Expression of GRX mRNA is very high in beta-cells compared with other studied tissues, and GRX protein expression is high in islets and in brain; 2) GRX and TRX are localized in distinct microdomains in the cytosol of beta-cells; and 3) microinjection of recombinant GRX potentiated effects of NADPH on exocytosis, whereas TRX antagonized the NADPH effect. We propose that the NADPH/GRX/TRX redox regulation mediates a novel signaling pathway of nutrient-induced insulin secretion.