We here examined the potential biological function of phosphoenolpyruvate carboxykinase 1 (PCK1) in angiogenesis. shRNA- or CRISPR-Cas9-induced PCK1 depletion potently inhibited endothelial cell proliferation, migration, sprouting, and tube formation, whereas ectopic PCK1 overexpression exerted opposite activity. In HUVECs, Gαi3 expression and Akt activation were decreased following PCK1 depletion, but were augmented by ectopic PCK1 overexpression. In vivo, retinal expression of PCK1 gradually increased from postnatal day 1 (P1) to P5. The intravitreous injection of endothelial-specific PCK1 shRNA adenovirus at P1 potently inhibited the radial extension of vascular plexus at P5. Conditional endothelial knockdown of PCK1 in adult mouse retina increased vascular leakage and the number of acellular capillaries while decreasing the number of RGCs in murine retinas. In diabetic retinopathy patients, PCK1 mRNA and protein levels were up-regulated in retinal tissues. Together, PCK1 is essential for angiogenesis possibly by mediating Gαi3 expression and Akt activation.