The glucose stimulation of insulin secretion by pancreatic β-cells depends on increased production of metabolic coupling factors, among which changes in NADPH and ROS (reactive oxygen species) may alter the glutathione redox state (E_GSH) and signal through changes in thiol oxidation. However, whether nutrients affect E_GSH in β-cell subcellular compartments is unknown. Using redox-sensitive GFP2 fused to glutaredoxin 1 and its mitochondria-targeted form, we studied the acute nutrient regulation of E_GSH in the cytosol/nucleus or the mitochondrial matrix of rat islet cells. These probes were mainly expressed in β-cells and reacted to low concentrations of exogenous H₂O₂ and menadione. Under control conditions, cytosolic/nuclear E_GSH was close to −300 mV and unaffected by glucose (from 0 to 30 mM). In comparison, mitochondrial E_GSH was less negative and rapidly regulated by glucose and other nutrients, ranging from −280 mV in the absence of glucose to −299 mV in 30 mM glucose. These changes were largely independent from changes in intracellular Ca²⁺ concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 (superoxide dismutase 2) and mitochondria-targeted catalase, but were inversely correlated with changes in NAD(P)H autofluorescence, suggesting that they indirectly resulted from increased NADPH availability rather than from changes in ROS concentration. Interestingly, the opposite regulation of mitochondrial E_GSH and NAD(P)H autofluorescence by glucose was also observed in human islets isolated from two donors. In conclusion, the present study demonstrates that glucose and other nutrients acutely reduce mitochondrial, but not cytosolic/nuclear, E_GSH in pancreatic β-cells under control conditions.