Objective: The aim of the study was to evaluate the effect of the RhoA/ROCK inhibitor Fasudil on retinal neovascularization (NV) in vivo and angiogenesis in vitro. Methods: C57BL/6 was used to establish an OIR model. First, RhoA/ROCK expression was first examined and compared between OIR and healthy controls. Then, we evaluated the effect of Fasudil on pathological retinal NV. Whole-mount retinal staining was performed. The percentage of NV area, the number of neovascular tufts (NVT), and branch points (BP) were quantified. Finally, human umbilical vein endothelial cells (HUVECs) were used to investigate the effect of Fasudil on angiogenesis. Results: Real-time PCR and Western blotting showed that ROCK expression in retinal tissue was statistically upregulated in OIR. Furthermore, we found that Fasudil attenuated the percentage of NV area, the number of NVT, and BP significantly. In addition, Fasudil could suppress the proliferation and migration of HUVECs induced by VEGF. Conclusions: RhoA/ROCK might be involved in the pathogenesis of OIR. And its inhibitor Fasudil could suppress retinal NV in vivo and angiogenesis in vitro. Fasudil may be a potential treatment strategy for retinal vascular diseases.