Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium such as angiogenesis or vessel repair in processes involving endothelial Nitric Oxide Synthase (eNOS) activation. On the other hand, one of the effector kinases that become activated in endothelial cells upon VEGF treatment is Protein Kinase D (PKD). We show herein that PKD uses eNOS as substrate leading to its activation with the concomitant increased ·NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or PDBu activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound-healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation as monitored through the determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a novel regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone.