Abstract: Objective To investigate whether human umbilical vein endothelial cells as a feeder layer was capable of supporting the growth of embryonic stem cells in vitro. Methods Human umbilical vein endothelial cells were isolated and cultured and then prepared as feeder cells after 3 passages. Alkaline posphatase activity (AKP) staining, stem cell surface marker test and karyotypes were conducted during different periods of cell culture. The suspension of stem cells cultured after 20 passages on endothelial cells were inoculated to the legs of severe combined immunodeficiency (SCID) mice subcutabeously to test the teratoma formation. Results E14.1 embryonic stem cells retained good colonies when they were cultured on endothelial cells for 3 passages and 8 passages. In addition, they expressed SSEA-1, Oct-4, and a membrane alkaline phosphatase to a high extent at passages 3 and 8. Embryonic stem cells cultured for 15 passages stably retaind a normal karyotype. Embryonic stem cells cultured on endothelial cells for 20 passages were inoculated into the hind leg of SCID mouse, teratomas containing three embryonic layers were recovered six weeks later. Conclusion Human umbilical vein endothelial cells would support effectively embryonic stem cells expansion, and provide a clinically safe method to expand ES cells for future clinical application.