Oxidative stress is one of the main contributors in the pathogenesis of diabetic retinopathy. The aim of this study is to investigate the effects of SS31 which is a mitochondria-targeted antioxidant peptide on the retinas of streptozotocin (STZ)-induced diabetic rats. Two weeks after induction of diabetes, SS31 (3 mg/kg) or the same volume of normal saline (N.S) was injected subcutaneously into the back of diabetic rats every day. Four months later, the integrity of inner blood retinal barrier (iBRB) was measured by Evans blue perfusion. The expression and distribution of claudin-5, occludin, acrolein, 8-OHdG and nitrotyrosine in the rat retinas were detected by immunofluorescent staining. Retinal ultrastructures were observed by transmission electron microscopy. The protein level of VEGFR2, Trx-2, Bcl-2, Bax, caspase-3, p53, and NF-κB in the rat retinas were assayed by western blot. Four months after subcutaneous injection, the diabetic rats treated with SS31 had better structures of retinal ganglion cells, thinner capillary basement membrane, less iBRB leakage, more uniform staining of claudin-5 and occludin in the retinal vessels, lower levels of acrolein, 8-OHdG, nitrotyrosine, Bax, caspase-3, p53, and NF-κB, and higher levels of Trx-2 and Bcl-2 in the retinas than those treated with N.S. In conclusion, SS31 could protect the retinal structures and inhibit the breakdown of iBRB by reducing oxidative damage, increasing Trx-2 and Bcl-2 expression, and decreasing p53, NF-κB, Bax, caspase-3, and VEGFR2 expression in the retinas of diabetic rats. SS31 could be a potential new treatment for diabetic retinopathy and other oxidative stress-related diseases.