Diabetes-induced oxidative stress is vital in initiating neuronal damage in the diabetic retina, leading to diabetic retinopathy (DR). This study investigates the possible effects of coumestrol (CMS) on streptozotocin (STZ)-induced DR. First, we established a rat model of DR by STZ injection and a cell model involving high-glucose (HG) exposure of human retinal microvascular endothelial cells (hRMECs). We characterized the expression patterns of oxidative stress indicators, pro-inflammatory cytokines, and pro-apoptotic proteins in hRMECs. Polymerase chain reaction showed sirtuin 1 (SIRT1) to be poorly expressed in the retinal tissues of STZ-treated rats and HG-exposed hRMECs, but its expression was upregulated upon treatment with CMS treatment. Furthermore, CMS treatment attenuated the STZ-induced pathologies such as oxidative stress, inflammation, and cell apoptosis. Consistent with the in vivo results, CMS activated the expression of SIRT1, thereby inhibiting oxidative stress, inflammation, and apoptosis of HG-treated hRMECs. From these findings, we concluded that CMS ameliorated DR by inhibiting inflammation, apoptosis and oxidative stress through activation of SIRT1.