Blood-brain barrier (BBB), which is formed by brain microvessel endothelial cells (BMECs), astrocytes and brain pericytes, selectively transports substances in the blood. Nutrients are actively taken up by the brain through various transporters on BMECs. In particular, docosahexaenoic acid (DHA) is most abundant nutrient in the brain and important for many neurophysiological functions. A major transporter for DHA at the BBB is the major facilitator superfamily domain containing protein 2a (MFSD2A), which is expressed exclusively in BMECs. Although brain pericytes regulate expression of MFSD2A on BMECs, its mechanism remains unclear. Here, we used non-contact cocultured BBB models consisting of primary cultures of rat brain endothelial cells (RBECs) and rat brain pericytes. After 1 and 3 days of coculture, expression levels of MFSD2A were evaluated by western blot analysis and immunofluorescent staining. The impact of brain pericytes on endothelial DHA uptake was evaluated by [14C] DHA radioactivity in RBECs cocultured with rat brain pericytes. In RBECs cocultured with rat brain pericyte for 3 days, MFSD2A protein expression in cell lysate and plasma membrane was significantly increased compared with RBEC monolayer. Brain pericytes significantly increased uptake of [14C] DHA by RBECs. Our findings suggested that brain pericytes promote DHA uptake by BMECs through increasing MFSD2A expression on plasma membrane of BMECs.