OBJECTIVE To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses. SAMPLE Jugular venous blood samples from 24 adult horses. PROCEDURES Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates. RESULTS Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry. CONCLUSIONS AND CLINICAL RELEVANCE ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.