Due to its essential roles in angiogenesis, Notch pathway has emerged as an attractive target for the treatment of pathologic angiogenesis. Although both activation and blockage of Notch signal can impede angiogenesis, activation of Notch signal may be more promising because it was shown that long-term Notch signal blockage resulted in vessel neoplasm. However, an in vivo deliverable Notch ligand with highly efficient Notch-activating capacity has not been developed. Among all the Notch ligands, Delta-like4 (Dll4) is specifically involved in angiogenesis. In this study, we generated a novel soluble Notch ligand hD4R, which consists of the Delta-Serrate-Lag-2 fragment of human Dll4 and an arginine-glycine-aspartate (RGD) motif targeting endothelial cells (ECs). We demonstrated that hD4R could bind to ECs through its RGD motif and effectively triggered Notch signaling in ECs. Further, we confirmed that hD4R could suppress angiogenesis in vitro as manifested by network formation assay and sprouting assay. More importantly, hD4R efficiently repressed neonatal retinal angiogenesis and laser-induced choroidal neovascularization (CNV) as well in vivo. In conclusion, we have developed an in vivo deliverable Notch ligand hD4R, which suppresses angiogenesis both in vitro and in vivo, thus providing a new approach to tackle excessive angiogenesis relevant disease such as CNV.