Since grape extract (GE) contains oligomeric proanthocyanidins and numerous polyphenols, dietary GE supplements may exert protective effects against various diseases. The present study investigated the pharmacological effects of GE derived from Chardonnay in vitro and in vivo. GE (100 µg/mL) completely inhibited tumor necrosis factor-α-induced endothelin-1, monocyte chemoattractant protein-1, interleukin-1β, and intercellular adhesion molecule-1 gene expression in cultured endothelial cells. GE also strongly stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) pathway. In the in vivo study, the effects of GE on ischemic acute kidney injury (AKI) were examined using male C57bl/6J wild-type and eNOS mice. Right nephrectomized mice were exposed to 45 min of ischemia in the left kidney and this was followed by reperfusion. Although renal functional parameters in AKI mice significantly increased 48 h after reperfusion, the administration of GE (0.1 and 1 mg/kg, intravenous (i.v.)) 5 min before ischemia dose-dependently improved post-ischemic renal dysfunction in wild-type mice. Renal histopathological studies on AKI mice revealed tubular necrosis, proteinaceous casts in tubuli, and medullary congestion. The administration of GE ameliorated this damage in wild-type mice, but not in eNOS mice. Furthermore, GE significantly restored decreases in the renal nitric oxide metabolite content due to ischemia in wild-type mice, but not in eNOS mice. Thus, eNOS is closely involved in the renoprotective effects of GE, strongly suggesting that GE supplements are useful as a prophylactic treatment for the development of ischemic AKI.