BACKGROUND He-Ying-Qing-Re Formula (HF) is a formula modified from "Si-Miao-Yong-An Decoction", a traditional Chinese medical classic emerged in the Qing dynasty and has been reported for treatment of vascular diseases. HF, containing 8 herbs, has been used in local hospital for decades as a complementary method for diabetic retinopathy (DR) with retinal vascular dysfunction. Clinical reports revealed HF could ameliorate vision defects, microaneurysms, hemorrhages and macular edema. The aim of this study is to investigate the anti-DR action of HF and its underlying mechanism experimentally. METHODS Chromatographic fingerprinting of HF and rodent model of DR were established; hypoglycemic effect of HF was measured by fasting, random blood glucose and glucose tolerance test; vascular degeneration was measured by retinal digestion; blood-retina-barrier (BRB) permeability was assessed with Evans Blue leakage assay. Advanced glycation end products (AGEs) were measured in vitro and in vivo level; Migration of retinal vascular endothelial cells were determined by wound healing and transwell chamber assays; permeability of endothelial monolayer was monitored with dextran transport. AGEs-related proteins and signaling were measured with immunoblotting and immunohistochemistry. RESULTS Chlorogenic acid, ferulic acid and arctin were identified as major components in HF; HF suppresses retinal vasculature degeneration and BRB permeability damage without significant inhibition on hyperglycemia; HF reduces in vitro and in vivo formation of AGEs and AGEs-induced migration as well as permeability of retinal vascular endothelial cells. Expression of tight junction proteins Zo-1 and Claudin-1 was increased while activation of AGEs receptor and downstream signaling Akt were suppressed upon HF treatment. CONCLUSIONS HF exhibits protective effect against diabetic retinopathy, which may be associated with inhibition on AGEs and recovery on endothelial dysfunction via modulation of tight junction and AGEs downstream signaling.