Diabetes increases oxidant stress and impairs endothelium‐dependent relaxation. We investigated whether the antioxidant 3′,4′‐dihydroxyflavonol (DiOHF) reduces superoxide (O2−) and preserves endothelial function in aortae from diabetic rats. In Sprague‐Dawley rats 8 weeks after streptozotocin (55 mg/kg iv) blood glucose was elevated (39.4±0.4 mmol/l) compared to citrate treated control (5.5±0.1 mmol/l, p<0.05) and, using lucigenin‐enhanced chemiluminescence, there was an increased aortic generation of O2− (control 670±101, diabetic 1535±249 units/mg dry weight, p<0.05). In aortic rings precontracted with U46619 ACh‐induced relaxation was impaired (Rmax control 79±2, diabetic 66±3%, p<0.01) whereas endothelium‐independent relaxation to SNP was unaffected. In the presence of DiOHF (10−4 M) there was a significant reduction in O2− generation (165±21 units/mg, p<0.01) and enhanced relaxation to ACh (Rmax 83±4%). 2 separate groups of rats (control and diabetic) were treated daily with DiOHF (5 mg/kg ip) for 7 days. DiOHF did not affect blood glucose (control + DiOHF 5.5±0.1 mmol/l, diabetic + DiOHF 38.8±0.5 mmol/l) but reduced superoxide generation in diabetic aortae (580±115 units/mg, p<0.05) and enhanced ACh‐induced relaxation (Rmax 80±4%, p<0.05). DiOHF, acutely or after 1 week treatment, reduces oxidant stress and preserves endothelium‐dependent relaxation in aortae from diabetic rats.