Inhibition of angiogenesis is an important therapeutic strategy for advanced stage prostate cancer (PCa). RhoA/Rho-associated protein kinases (ROCK) are key regulators of the cytoskeleton and have been implicated in PCa angiogenesis. We investigated the anti-angiogenic effects of fasudil, a ROCK inhibitor, on PCa-induced angiogenesis in vitro. Proliferation of PCa-conditioned human umbilical vein endothelial cells (HUVECs) was assessed using a bromodeoxyuridine (BrdU) assay, and migration was assessed with a wound healing assay. In vitro angiogenesis of PCa-conditioned HUVECs was evaluated by tube formation and a spheroid sprouting assay. Fasudil inhibited PCa-induced endothelial cell proliferation at a concentration of 100 µM, and also decreased PCa-induced endothelial cell migration at a concentration of 30 µM. In the in vitro angiogenesis assay, fasudil exerted a more significant effect. Tube formation was significantly inhibited at fasudil concentrations exceeding 3 µM, and spheroid sprouts were significantly thinner and shorter (at fasudil concentrations of 10 and 30 µM, respectively). Western blotting results showed that expression of phosphorylated myosin phosphatase target subunit 1 (MYPT-1) was significantly lower after fasudil treatment, confirming that fasudil inhibited ROCK activity in these model systems. These data suggest that fasudil may be a useful anti-angiogenic agent for PCa.