We examined the effects of reduced Na⁺/K⁺-ATPase activity on mitochondrial ATP production and insulin release from rat islets. Ouabain, an inhibitor of Na⁺/K⁺-ATPase, augmented 16.7 mmol/l glucose–induced insulin release in the early period but suppressed it after a delay of 20–30 min. Unexpectedly, the ATP content in an islet decreases in the presence of 16.7 mmol/l glucose when Na⁺/K⁺-ATPase activity is diminished by ouabain, despite the reduced consumption of ATP by the enzyme. Ouabain also suppressed the increment of ATP content produced by glucose even in Ca²⁺-depleted or Na⁺-depleted conditions. That mitochondrial membrane hyperpolarization and O₂ consumption in islets exposed to 16.7 mmol/l glucose were suppressed by ouabain indicates that the glycoside inhibits mitochondrial respiration but does not produce uncoupling. Ouabain induced mitochondrial reactive oxygen species (ROS) production that was blocked by myxothiazol, an inhibitor of site III of the mitochondrial respiratory chain. An antioxidant, α-tocopherol, also blocked ouabain-induced ROS production as well as the suppressive effect of ouabain on ATP production and insulin release. However, ouabain did not directly affect the mitochondrial ATP production originating from succinate and ADP. These results indicate that ouabain suppresses mitochondrial ATP production by generating ROS via transduction, independently of the intracellular cationic alternation that may account in part for the suppressive effect on insulin secretion.