Endothelial cells (ECs) apoptosis induced by hyperglycemia is intimately involved in the pathophysiology of diabetes and its complication. Although PGC-1α is known for its role in glucose metabolism, its role in ECs injury caused by high glucose milieu is still unclear. Therefore, this study aims to investigate whether PGC-1α participates in ECs apoptosis under high glucose condition. Human umbilical vein endothelial cells (HUVECs) were down-regulated PGC-1α expression by adenovirus-mediated PGC-1α specific siRNA (Ad-shPGC-1α) and exposed to high glucose. Cell viability, apoptosis, mitochondrial membrane permeability, apoptotic marker, reactive oxygen species (ROS), and expression of PGC-1α and VDAC isoforms were studied. Our results showed that high glucose-induced cell apoptosis was associated with an obvious decrease in PGC-1α expression. Lack of PGC-1α exacerbated high glucose-induced cell apoptosis, inner mitochondrial membrane permeabilization, mitochondrial cytochrome c release into cytoplasm and caspases activation; while further decreased cell viability and mitochondrial membrane potential. Analysis of apoptotic markers (Bcl-2, Bax), intracellular ROS and endoplasmic reticulum stress revealed that these mechanisms were not accounted for the effects of Ad-shPGC-1α on apoptosis. However, we found silencing PGC-1α further increased high glucose-induced VDAC1 expression. The pharmacological inhibition of VDAC1 with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited the increased apoptosis in high glucose-treated PGC-1α knockdown cells. These findings strongly suggest that PGC-1α defect is one of the major mechanisms for ECs apoptosis under high glucose condition, and provide a novel strategy to prevent endothelial dysfunction in diabetes.