Objective:To develop an easy and reduplicate method for the isolation and primary culture of vascular endothelial cells of I-资B kinase ε(IKKε) knock-out(KO) mouse. Method: The aorta was obtained from IKKε KO mouse in the super-clean bench,with its adventitia and adipose tissue cleared carefully. Then the vessel was split longitudinally and cut into 1 mm × 1 mm explants. The explants were placed on culture dish with the lining endothelium side down and DMEM containing endothelial cell growth supplement(ECGS) was added into the culture dish. The morphological characteristics of the cells were observed with the inverted microscope and the cells were evidenced by immunofluorescence with marker Ⅷ factor. Results: Endothelial cells migrating out of the aortic intima can be seen at the edge after 72 hours,forming confluent monolayer 12~14 days later. Immunofluorescence showed the characteristic positive reaction of Ⅷ factor in the cytoplasm of the cells. Conclusion: High purity,solid structure and sound function IKKε KO mouse endothelial cells can be obtained from this method.