Abstract: Objective To establish a reliable method for the isolation and culture of port wine stain （PWS） endothelial cells, and to observe their characteristics in vitro. Methods Tissue specimens were resected from patients with PWS, and digested with collagenase to obtain single cell suspension. Endothelial cells were isolated using anti-CD31 coated Dynabeads. The CD31+ cells were inoculated into fibronectin coated culture-dishes. Then, morphological features were observed and growth curve was drawn for these cells. The ability of these cells to scavenge Dil-acetylated-low density lipoprotein （LDL） was assessed. Also, immunofluorescent labeling was used to measure the expressions of von Willebrand factor （vWf） and CD31 in these cells. Results CD31+ cells were isolated from PWS tissues. Cells, which had a spindle appearance, started to adhere within 6 hours on the fibronectin coated culture-dishes; they reached confluence and demonstrated a typical endothelial cobblestone morphology on day 7 in culture. The cells stained positive for CD31 and vWF, and could scavenge Dil-acetylated-LDL. Conclusion A method has been established for the isolation and culture of PWS endothelial cells in this study.