Abstract: Objective To explore the role of the interaction between glycogen synthase kinase- 3β (GSK- 3β) and endoplasmic reticulum stress (ERS) in the high glucose (HG)-induced injury in human umbilical vein endothelial cells (HUVECs). Methods HUVECs treated with 40 mmol/L glucose for 24 h were examined for expression levels of GSK-3β, GRP78, CHOP and cleaved caspase-3 protein using Western blotting. The cell viability was examined using CCK-8 assay and cell apoptosis was detected with Hoechst 33258 nuclear staining and photofluorography. The intracellular level of reactive oxygen species (ROS) was measured with dichlorfluoresein staining and photofluorography. Mitochondrial membrane potential (MMP) was tested by rhodamine 123 (Rh123) staining and photofluorography. Results Treatment of HUVECs with 40 μmol/L glucose for 3-24 h activated GSK-3β in a time-dependent manner, leading to significantly down-regulated expression of phosphorylated (p)-GSK- 3β (P<0.05). HG exposure of the cells for 1-24 h induced ERS, evidenced by time-dependently up-regulated expression of GRP78 and CHOP (P<0.05). LiCl, an inhibitor of GSK-3β, attenuated HG-induced ERS and significantly lowered the expression levels of GRP78 and CHOP (P<0.01). 4-PBA, an inhibitor of ERS, obviously ameliorated the activation of GSK-3β by HG as shown by the increase in p-GSK-3β expression level (P<0.01). HG exposure for 24 h induced obvious injuries in HUVECs, which exhibited decreased cell viability, increased cell apoptosis, increased expression of cleaved caspase-3 and ROS generation, and loss of MMP. Pretreatment of the cells with LiCl or 4-PBA for 60 min before HG exposure significantly lessened the cell injuries (P<0.01). Conclusion Interactions between GSK-3β and ERS occur in HUVECs exposed to HG and participate in HG-induced cell injuries.