Objective To establ ish an efficient and stable culture method of human umbil ical vein endothel ial cells (HUVECs) in vitro so as to provide good source of seed cells for tissue engineered vascular grafts and for precl inical research. Methods The umbil ical cords were harvested from full-term normal delivered neonates, which were perfused with0.1% collagenase II by self-made needle and were digested at 37 and 5% CO2 humidified incubator. The HUVECs were cultured in endothel ial culture medium (ECM) containing 5% fetal bovine serum (FBS) and 1% endothel ial cell growth factor (ECGS). HE staining of the umbil ical cords before and after digestion was used to observe the detachment of HUVECs, flow cytometry to detect the purity of primary HUVECs, and inverted phase contrast microscope to observe the morphology of the cultured HUVECs. The growth of the 3rd passage cells was measured by MTT assay; immunocytochemical technique and matrigelbased capillary-l ike tube formation assay were carried out to identify the function of HUVECs. Results After digestion of 0.1% collagenase II, marked HUVECs detachment was observed with complete digestion. The purity of the HUVECs was 99.56% by digestion of 0.1% collagenase II at 37 and 5% CO2 humidified incubator for 15 minutes. Primary HUVECs showed a cobblestone or pitching stone-l ike appearance in vitro, forming a confluent monolayer cells after 2-3 days of culture. MTT assay demonstrated that HUVECs showed the fastest growth speed at 3 to 4 days, and showed growth of cell fusion at about 5 days. Immunocytochemistry showed that HUVECs highly expressed endothel ial marker factor VIII. Matrigel based capillary-l ike tube formation assay showed that it could form endothel ial-l ike tube structures after 24 hours of culture. Conclusion Using improved method and ECM could obtain high quantity and high qual ity primary HUVECs, which might be a kind of promising seed cells for tissue engineering and precl inical research.