Angiogenesis plays a pivotal role in the formation and homeostasis of blood vessels during embryonic and postnatal development, but its regulatory mechanism has not been fully understood.In this study, we identified miR-342-5p as a novel modulator in endothelial cells (ECs) to regulate angiogenesis, miR-342-5p was differentially expressed in ECs from quiescent and angiogenic vessels.The expression of miR-342-5p was regulated by VEGFR signaling, Notch signaling, and TGF-β, all of which are critically involved in angiogenesis.We found that transfection of a miR-342-5p mimic inhibited the proliferation and promoted the migration of ECs in vitro.Transfection of miR-342-5p also inhibited aggregation of ECs into networks.Culture of aortic rings and a beads-based sprouting assay demonstrated that miR-342-5p overexpression reduced the length of angiogenic sprouts, although the number of sprouts might be increased.Furthermore, miR-342-5p mimic inhibited angiogenesis of retinal vasculature of P5 newborn mice and laser-induced CNV in adult mice.At the molecular level, miR-342-5p inhibited Akt phosphorylation and ERK phosphorylation in ECs induced by VEGF.Interestingly, miR-342-5p also regulated TGF-β signaling through targeting Endoglin.Downregulation of endoglin by miR-342-5p reduced the phosphorylation of SMAD1/5 while SMAD2/3 phosphorylation was untouched, leading to reduced endothelial markers and increased mesenchymal markers, a property of increased endo-MT.Lastly, we found that miR-342-5p collaborated to regulate EC migration.These data clarified miR-342-5p as a novel coordinator among critical angiogenic pathways.