Endothelial cell injury and dysfunction caused by reactive oxygen species (ROS) are implicated in the pathogenesis of vascular diseases. ROS are generated and hypoxanthine is degraded by xanthine oxidase. Smoking and alcohol consumption are associated with an increased level of hypoxanthine. We aimed to study the direct role of hypoxanthine in endothelial dysfunction in human umbilical vascular endothelial cells (HUVECs). Hypoxanthine induced cell death and production of ROS. Furthermore, hypoxanthine induced apoptosis through regulation of protein expression related to apoptosis. When cells were pretreated with N-acetylcysteine or a pancaspase inhibitor (Z-VAD-fmk) and stimulated with hypoxanthine, Z-VAD-fmk and N-acetylcysteine prevented hypoxanthine-induced apoptosis by inhibiting the ROS production and caspase pathway. Thus, an increased extracellular concentration of hypoxanthine induces endothelial dysfunction through ROS production and regulates expression of apoptosis-related proteins in HUVECs. These effects are expected to be associated with some vascular diseases.