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Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. It plays a primary role in a selective diffusion barrier at the level of the cerebral microvascular endothelium. Here, we have developed and characterized an in vitro model of the blood-brain barrier by seeding primary brain microvascular endothelial cells (BMECs) and astrocyte cells (ACs) from sheep on a polyester Transwell cell culture membrane with 0.4-μm pores, and conducted transepithelial electrical resistance measurements (TEER), leakage tests and assays for the permeability of horseradish peroxidase (HRP). The results of TEER and 4-h leakage assay showed that the new syngeneic BBB model had a characteristic of BBB in vivo. The HRP permeability test showed that the BBB model had good barrier function. Results showed that our model may be a valuable tool in the study of the mechanisms of brain invasion mediated by internalins of Listeria monocytogenes.