Brain microvascular endothelial cell(BMVEC)is an essential component of the brain blood barrier(BBB). It has been known that the dysfunction of BBB plays critical roles in the pathological process of a variety of neurological disorders. The primary cultured BMVECs were widely used in the study of the functions and the regulation mechanisms of the BBB. Here,it is reported that a simple and effective method of isolating and growing of BMVEC established upon density gradient centrifugation and enzyme digestion. The purity of the cultured cells was higher than 95% determined by CD31 immunofluorescence staining. The features of the cells were further confirmed by positive staining of VE-cadherin and ZO-1,the characteristics of BMVECs. Our method may provide a good approach for the study of BBB in vitro.