The aim of this study was to investigate the protective effect of 5-HMF on human umbilical vein endothelial cells (HUVECs) injured by high glucose in vitro, and the mechanism underlying this process. Our results demonstrated that high glucose-induced oxidative stress in HUVECs was mainly mediated through activation of reactive oxygen species (ROS), Jun N-kinase 2/3 (JNK2/3) and plasma interleukin-8 (IL-8), and inactivation of phosphorylated protein kinase B (P-Akt). Treatment of HUVECs with media containing high glucose (4.5%) in the presence of 5-HMF (100, 200 and 400 μM) resulted in significant inhibition of high glucose-induced oxidative stress and expression of JNK1 and JNK2/3. Furthermore, 5-HMF rapidly inhibited high glucose-induced activation of IL-8, a downstream activator of P-Akt. Diabetes mellitus can cause a wide variety of vascular complications and high glucose can induce vascular endothelial cell apoptosis. Free radicals are formed disproportionately in diabetes by glucose oxidation. The finding of this study highlights the pharmacological application of 5-HMF for preventing cardiovascular and diabetes mellitus diseases, and provides the theoretical basis for further development of a Cornus officinalis agent for diabetes-associated vascular diseases.