Renal ischemia-reperfusion (I/R) results in vascular dysfunction characterized by a reduced endothelium-dependent vasodilatation and subsequently impaired blood flow. In this study we investigated the role of Rho kinase in eNOS mediated regulation of renal blood flow and vasomotor tone in renal I/R. Male Wistar rats were subjected to 60 minutes bilateral clamping of the renal arteries or sham procedure. One hour before clamping, the Rho kinase inhibitor Y27632 (1 mg/kg) was intravenously infused. After I/R renal blood flow was measured using fluorescent microspheres. I/R resulted in a 62% decrease in renal blood flow. In contrast, the blood flow decrease in the group treated with the Rho kinase inhibitor (YI/R) was prevented. Endothelium-dependent vasodilatation of renal arcuate arteries to acetylcholine (Ach) was measured ex vivo in a pressure myograph. These experiments demonstrated that the in vivo treatment with the Rho kinase inhibitor prevented the decrease in NO-mediated vasodilator response. In addition, after I/R renal interlobar arteries showed a decrease in phosphorylated eNOS and vasodilator-stimulated phosphoprotein (VASP), a marker for bioactive NO, which was attenuated by in vivo Rho kinase inhibition. These findings indicate that in vivo inhibition of Rho kinase in renal I/R preserves renal blood flow by improving eNOS function.