Abstract Objectives Diabetic retinopathy (DR) is a complication of diabetes, caused by high blood glucose levels damaging to blood vessels in the retina. It can leading cause of blindness. Nobiletin is a polymethoxyflavone present in citrus fruit and peels, it has anti-cancer and anti-inflammatory effects. This study investigated the protective effects of nobiletin on retinal blood-retinal barrier (BRB) breakdown in high glucose-exposed human retinal endothelial cells and in db/db mouse eyes. Methods Human retinal microvascular endothelial cell (HRMVEC) were incubated in media exposed to 33 mM glucose in the absence and presence of 1–20 μM nobiletin up to 5 d. Antibodies ZO-1, MMP2, Bax and Bcl2 were used for western blot analysis. The in vivo animal model employed db/db mice orally administrated with 10 mg/kg of nobiletin. Results Non-toxic nobiletin declined the expression of ZO-1 which is retinal tight junction protein and Bcl2 expression in high glucose stimulation. when treated with nobiletin promoted the expression of ZO-1 and Bcl2 in HRMVEC cell. Also, of MMP2 and Bax was up-regulated by high glucose stimulation and nobiletin down-regulated high glucose induced expression of the MMP2 and Bax. In in vivo study, oral administration of 10 mg/kg nobiletin protected the retinal endothelial microvascular through recovering ZO-1 and Bcl2 expression as control. Also, nobiletin reduced tissue expressions of MMP2 and Bax. Conclusions These results demonstrated that nobiletin may be a potent retinoprotective agent to prevent diabetes-associated microvascular abnormalities and BRB breakdown in retinal microvascular functions leading to retinal failure. Funding Sources This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (2017R1A6A3A04011473).