The angiogenesis induced by tumor cells is essential for metastasis of hepatocellular carcinoma. Available information suggests that RhoC participates in angiogenesis through regulation of vascular endothelial growth factor expression in tumor cells. For its broad functions in cell migration and cytoskeletal organization, we hypothesized that RhoC regulating angiogenesis does not exclusively depend on regulation of vascular endothelial growth factor expression. To address this question, in the present study, we used a retroviral small interfering RNA approach to selectively knockdown the expression of RhoC in a neovascularization model in vivo and in vitro. Our present results indicate that RhoC is the downstream regulator of vascular endothelial growth factor in endothelial cells and is essential for angiogenesis induced by vascular endothelial growth factor, notwithstanding that RhoC regulates the expression of vascular endothelial growth factor in tumor cells. Furthermore, we show that knockdown of RhoC is associated with the inhibition of invasion and migration but not apoptosis of endothelial cells. Knockdown of RhoC results in inhibition of endothelial cell organization through restraining the reorganization of F-actin filaments, which represses endothelial cell network and sprout formation. In conclusion, our results demonstrate that knockdown of RhoC inhibits angiogenesis induced by tumor cells not only through effecting the release of vascular endothelial growth factor, but also through inhibiting endothelial cell migration and organization, which implies that it blocks tumor metastasis by specifically inhibiting RhoC in endothelial cells.