Vascular endothelial growth factor (VEGF)-VEGFR2 pathway is a key regulator of retinal angiogenesis. We have previously showed that ankyrin repeat and FYVE domain containing 1 (ANKFY1), a Rab5-GTP-interacting protein, regulate the formation of endothelial cell tube-formation. However, in retinal endothelial cells, the physiological functions of ANKFY1 are unclear. Here, we investigated the effects of ANKFY1 depletion on retinal endothelial cells. The cell proliferation and migration were evaluated in ANKFY1 knockdown human retinal microvascular endothelial cells (HRMECs). To evaluate the effects of ANKFY1 depletion on expression levels of pro-angiogenic receptors and intracellular signaling, cells were analyzed by Western blot or qRT-PCR. Depletion of ANKFY1 decreased the endothelial cell proliferation and migration in the presence or absence of VEGF. Western blot analysis showed that the cell surface level of VEGFR2 was decreased by ANKFY1 knockdown. Furthermore, the Akt/eNOS pathway was downregulated in ANKFY1 knockdown HRMECs. These findings indicate that ANKFY1 is a novel therapeutic target for ocular angiogenesis.