GSE (grape seed extract) has been shown to exhibit protective effects against cardiovascular events and atherosclerosis, although the underlying molecular mechanisms of action are unknown. Herein, we assessed the ability of GSE to enhance eNOS (endothelial nitric oxide synthase) expression and NO (nitric oxide) production in H2O2 (hydrogen peroxide)-treated HUVECs (human umbilical vein endothelial cells). GSE enhanced eNOS expression and NO release in H2O2-treated cells in a dose-dependent manner. GSE inhibited intracellular ROS (reactive oxygen species) and reduced intracellular calcium in a dose-dependent manner in H2O2-treated cells, as shown by confocal microscopy. ROS was inhibited in cells pretreated with 5.0 μM GSE, 2.0 μM TG (thapsigargin) and 20.0 μM 2-APB (2-aminoethoxydiphenyl borate) instead of 0.25 μM extracellular calcium. In addition, GSE enhanced eNOS expression and reduced ROS production via increasing p-AKT (AKT phosphorylation) with high extracellular calcium (13 mM). In conclusion, GSE protected against endothelial injury by up-regulation of eNOS and NO expression via inhibiting InsP3Rs (inositol 1,4,5-trisphosphate receptors)-mediated intracellular excessive calcium release and by activating p-AKT in endothelial cells.