Venous endothelial cell apoptosis can be induced by endoplasmic reticulum (ER) stress, thus serving an important role in the formation of deep venous thrombosis. X-box binding protein 1 (XBP1) is a protein associated with ER. The present study aimed to explore the function of XBP1/C/EBP homologous protein (CHOP) pathway in the process of endothelial cell apoptosis under hyperglycemia. Small interfering (si)RNAs targeting XBP1 and CHOP were designed to downregulate the expression of XBP1 and CHOP in human umbilical vein endothelial cell, respectively. Flow cytometry was used to determine cell apoptosis. The expression of XBP1, glucose-regulated protein 78 (GRP78), CHOP, Puma, cleaved caspase-3 and Cytochrome c was evaluated by western blotting. There were seven groups of cells that were used in the present study: i) Control (5.5 mM D-glucose); ii) hypertonic (hypertonic control, 27.8 mM mannitol and 5.5 mM D-glucose); iii) 16.7 mM D-glucose; iv) 33.3 mM D-glucose; v) 33.3 mM + NC (33.3 mM D-glucose incubated with NC); vi) 33.3 mM + si-XBP1 (33.3 mM D-glucose incubated with siRNA against XBP1); and vii) 33.3 mM + si-CHOP (33.3 mM D-glucose incubated with siRNA against CHOP). Compared with the control, the apoptosis rate of human umbilical vein endothelial cells (HUVECs) increased greatly with the increase in the concentration of D-glucose. Compared with the 33.3 mM D-glucose group, the HUVECs incubated with 33.3 mM D-glucose and si-XBP1 or 33.3 mM D-glucose and si-CHOP demonstrated a significantly lower apoptosis rate. Compared with the control, XBP1, GRP78, CHOP, Puma, cleaved caspase-3 and cytochrome c were significantly upregulated in the hypertonic, 16.7 mM D-glucose, 33.3 mM D-glucose and 33.3 mM + negative control (NC) groups. Compared with the 33.3 mM D-glucose group, the expression levels of XBP1, GRP78, CHOP, Puma, cleaved caspase-3 and cytochrome c in the 33.3 mM + si-XBP1 or 33.3 mM + si-CHOP groups significantly decreased. High dosage of glucose induced endothelial cell apoptosis by promoting the expression of apoptotic proteins by activating endoplasmic reticulum stress. XBP1/CHOP may be a potential target for the treatment of deep vein thrombosis as one of the key pathways regulating ERS by regulating apoptosis of endothelial cells.