Recent studies suggest that oxidative stress plays a major role on retinal dysfunction in diabetes. The Nrf2/ARE antioxidant pathway has been found to be adaptively activated to counteract increased oxidative stress. However, the activation of Nrf2/ARE pathway has not been studied in the retina of the genetic type 2 diabetic animal model, db/db mice. The present study is to investigate the oxidative status and the activation of Nrf2/ARE antioxidant pathway in retinas of db/db mice. Male db/db C57BLKS/J mice aged at 8, 12, and 20 wks were used in this study. Age-matched male heterozygous db/m littermates were used as control. Animals were enucleated under anesthesia induced with ketamine and xylazine (80 mg/kg+10 mg/kg; intraperitoneally) and isolated retinas were prepared for reactive oxygen species (ROS) detection, immunohistochemistry, and western blotting analysis. All experiments were performed in accordance with Peking University guidelines for animal research and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. ROS generation was detected by dihydroethidium (DHE) assay, the expression of NADPH oxdase-4 (Nox4) and heme oxygenase-1 (HO-1) were examined by immunohistochemistry and western blotting. ROS generation in the retinas of db/db mice increased at 8 wks and continued to progress at 20 wks of ages, compared to age-matched db/m mice (8 wks: dm: 9.67±0.84; db: 22.71±4.74, p<0.01, n=5; 20 wks: dm: 8.11±0.56; db: 21.13±3.71, p<0.01, n=5). However, the increased expression of Nox4 was only observed in the retina of 8 wks (dm: 10.80±1.23; db:17.10±5.37, p<0.01, n=5), but not 20 wks db/db mice compared to age-matched db/m mice. Meanwhile, in comparison with age-matched db/m mice, the expression of HO-1, an Nrf2-mediated antioxidant, was also increased in the retinas of db/db mice aged at 8 wks (dm: 9.68±1.17; db: 17.35±1.13, p<0.001, n=5), but not at 20 wks. In conclusion,the antioxidant pathway was adaptively activated to counteract hyperglycemia-induced oxidative stress to maintain the redox status in early stages of the diabetic retina (8 wk). As diabetes developed, the antioxidant capacity was damaged by the continuous hyperglycemia and oxidative stress.