Endothelial cell dysfunction is considered to be one of the major causes of vascular complications in diabetes. Polyphenols are known as potent antioxidants that can contribute to the prevention of diabetes. Corn silk has been reported to contain polyphenols and has been used in folk medicine in China for the treatment of diabetes. The present study aims to investigate the potential protective role of the phenolic-rich fraction of corn silk (PRF) against injuries to vascular endothelial cells under high glucose conditions in vitro and in vivo. The protective effect of PRF from high glucose toxicity was investigated using human umbilical vein endothelial cells (HUVECs). The protective effect of PRF was subsequently evaluated by using in vivo methods in streptozotocin (STZ)-induced diabetic rats. Results showed that the PRF significantly reduced the cytotoxicity of glucose by restoring cell viability in a dose-dependent manner. PRF was also able to prevent the histological changes in the aorta of STZ-induced diabetic rats. Results suggested that PRF might have a beneficial effect on diabetic patients and may help to prevent the development and progression of diabetic complications such as diabetic nephropathy and atherosclerosis.