Endostatin (ES) was reported to stimulate apoptosis in endothelial cells, but the exact mechanism remains controversial.In the present study, we tried to eiucidate the mechanism of ES-induced endothelial cell apoptosis.Our results indicate that ES induces cytochrome c release and caspase-9 activation in human microvascular endothelial cells (HMECs) at the concentration of 1 μM for 24 hours, which initiate the apoptosis process.Further ATP production, mitochondrial membrane potential, and tubule formation assays showed that ES promotes the mitochondrial permeability transition pore (mPrP) opening via voltage dependent anion channel 1 (VDAC1), a major component of mitochondria outer membrane pore.Knocking down VDAC 1 by siRNA attenuates ES-induced apoptosis, while over-expression of VDAC 1 enhances the sensitivity of endothelial cells to ES.Moreover, we reveal that ES induces the reduction of hexokinase 2 (HK2), which in turn promotes VDAC1 phosphorylation and accumulation.Data from 2D eiectrophoresis, immunoprecipitation, mPTP opening and caspase-3 activation assays indicates that two serine residues of VDAC 1, Ser-12 and Ser-103, can modulate VDAC 1 protein level and thus the sensitivity to apoptosis stimuli.Based on these findings, we thereby conclude that VDAC 1 plays a vital role in modulating ES-induced endothelial cell apoptosis.