Vascular endothelial growth factor VEGF is an endothelial specific growth factor that activates the small GTPase RhoA. While the role of RhoA for VEGF-driven endothelial migration and angiogenesis has been studied in detail, the function of its target proteins, the Rho dependent kinases ROCK I and II, are controversially discussed. Using the mouse model of oxygen-induced proliferative retinopathy, ROCK I/II inhibition by H1152 resulted in increased angiogenesis. This enhanced angiogenesis, however, was completely blocked by the VEGF-receptor antagonist PTK/ZK. Loss-of-function experiments in endothelial cells revealed that inhibition of ROCK I/II using the pharmacological inhibitor H1152 as well as ROCK I/II specific siRNAs resulted in a rise of VEGF-driven sprouting angiogenesis. These functional data were biochemically substantiated by showing an enhanced VEGF-receptor KDR phosphorylation and ERK1/2 activation after inhibition of ROCK I/II. Thus our data identify that the inhibition of Rho dependent kinases ROCK I/II activates angiogenesis both, in vitro and in vivo.