Diabetic cardiomyopathy is associated with an increase in oxidative stress. However, antioxidant therapy has shown a limited capacity to mitigate disease pathology. The molecular mechanisms responsible for the modulation of reactive oxygen species (ROS) production and clearance must be better defined. The objective of this study was to determine how insulin affects superoxide radical (O2•-) levels. O2•- production was evaluated in adult cardiomyocytes isolated from control and Akita (type 1 diabetic) mice by spin-trapping electron paramagnetic resonance spectroscopy. We found that the basal rates of O2•- production were comparable in control and Akita cardiomyocytes. However, culturing cardiomyocytes without insulin resulted in a significant increase in O2•- production only in the Akita group. In contrast, O2•- production was unaffected by high glucose and/or fatty acid supplementation. The increase in O2•- was due in part to a decrease in superoxide dismutase (SOD) activity. The PI3K inhibitor, LY294002, decreased Akita SOD activity when insulin was present, indicating that the modulation of antioxidant activity is through insulin signaling. The effect of insulin on mitochondrial O2•- production was evaluated in Akita mice that underwent a 1-week treatment of insulin. Mitochondria isolated from insulin-treated Akita mice produced less O2•- than vehicle-treated diabetic mice. Quantitative proteomics was performed on whole heart homogenates to determine how insulin affects antioxidant protein expression. Of 29 antioxidant enzymes quantified, thioredoxin 1 was the only one that was significantly enhanced by insulin treatment. In vitro analysis of thioredoxin 1 revealed a previously undescribed capacity of the enzyme to directly scavenge O2•-. These findings demonstrate that insulin has a role in mitigating cardiac oxidative stress in diabetes via regulation of endogenous antioxidant activity. Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.