Cardiovascular complications of diabetes mellitus involve oxidative stress and profound changes in reduced glutathione (GSH), an essential tripeptide that controls many redox-sensitive cell functions. This study examined regulation of GSH by insulin to identify mechanisms controlling cardiac redox state and to define the functional impact of GSH depletion. GSH was measured by fluorescence microscopy in ventricular myocytes isolated from Sprague Dawley rats made diabetic by streptozotocin, while video and confocal microscopy were used to measure mechanical properties and Ca²⁺ transients, respectively. Spectrophotometric assays of tissue extracts were also done to measure the activities of enzymes that control GSH levels. Four weeks after injection of streptozotocin, mean [GSH] in isolated diabetic rat myocytes was 36% less than control which correlated with decreased activities of two major enzymes regulating GSH levels: glutathione reductase and -glutamylcysteine synthetase. Treatment of diabetic rat myocytes with insulin normalized [GSH] after a delay of 3-4 h. A more rapid but transient up-regulation of [GSH] occurred in myocytes treated with dichloroacetate, an activator of pyruvate dehydrogenase. Inhibitor experiments indicated that insulin normalized [GSH] via the pentose pathway and -glutamylcysteine synthetase, although the basal activity of glucose-6-phosphate dehydrogenase was not different between diabetic and control hearts. Diabetic rat myocytes were characterized by significant mechanical dysfunction that correlated with diminished and prolonged Ca²⁺ transients. This phenotype was reversed by in vitro treatment with insulin and also by exogenous GSH or N-acetylcysteine, a precursor of GSH. Our data suggest that insulin regulates GSH through pathways involving de novo GSH synthesis and reduction of its oxidized form. It is proposed that a key function of glucose metabolism in heart is to supply reducing equivalents required to maintain adequate GSH levels for the redox control of Ca²⁺ handling proteins and contraction.