Diabetic retinopathy is one of the most common microvascular complications of diabetes. Inhibition of histone deacetylase 3 (Hdac3) was proved to be a successful way to ameliorate central nervous system injury and vision problem in glaucoma mouse model. However, its role in diabetic retinopathy remains largely unknown. Eight-week-old C57BL/6J mice were intraperitoneally injected with 50 mg streptozotocin for consecutive 5 days to induce diabetes. After one-week, diabetic mice were selected and treated with Hdac3 inhibitor, RGFP966, once every three days for consecutive 12 weeks. It was found that RGFP966 could decrease the mRNA and protein expression of Hdac3. It significantly thickened diabetic retinopathy-reduced retinal thickness without affecting fasting blood glucose. It also decreased diabetic retinopathy-activated oxidative stress and cell apoptosis. Moreover, diabetic retinopathy mice displayed an increased expression of vascular endothelial growth factor, and a decreased expression of glial fibrillary acidic protein, both of which were partially restored by RGFP966 treatment. Mechanically, RGFP966 decreased the expression of NADPH oxidase 2 (Nox2), while increased the expression of superoxide dismutase 2 (Sod2) in diabetic retinopathy mice. In conclusion, RGFP966 significantly reduces oxidative stress, inflammation, and cell apoptosis in the retina of streptozotocin-induced diabetic mice, which may be associated with its modulation on Nox2 and Sod2 expression.