〔Abstract〕 Objective To isolate, culture and identify rat femoral artery endothelial cellsin vitro, and to provide anin vitrocell model for pathological and physiological studies. Methods The rat femoral artery blood vessels were taken aseptically, and rat femoral artery endothelial cells were isolated by collagenase digestion; the purity of endothelial cells was detected by flow cytometry; CD31 positive cells were sorted out by flow cytometry, and endothelial cells were identified by laser confocal; the proliferation ability of endothelial cells was detected by CCK-8 method and high-content cell imaging method, and their migration and tube function were detected by Transwell method and Matrigel gel method. Results The primary endothelial cells of the femoral artery vascular obtained by collagenase digestion and separation had covered more than 2/3 of the area of the culture flask at 14-16 d, and the cells were typical paving stones.The purity was 38.34%, and the sorted cells were identified by CD31 fluorescence staining. The cells were all CD31 positive; PGE2can significantly upregulate the proliferation, migration, and tube formation of CD31 positive endothelial cells. Conclusion In this study, a simple method is used to successfully isolate the primary endothelial cells of femoral arteries, providing anin vitrocell model for studying the function of primary endothelial cells derived from relatively small blood vessels.