The blood brain barrier (BBB) is a physical and metabolic barrier, composed of multicellular unit, separating the central nervous system from blood circulation. One of BBB’s roles is to protect the brain of potential cytotoxic compounds. This protection is ensured by tight junction proteins (TJs), which create a physical blockade to paracellular diffusion, and by efflux transporters proteins like P-glycoprotein (Pgp) or multidrug resistance proteins (MRPs) (transendothelial pathway). These proteins acted as a barrier system by pumping compounds out of endothelial cells. The structural and functional integrity of transporters and TJs are neccesary for an intact BBB. Repeated hypoxia linked to some neurological disorders, like obstructive sleep apnea syndrome, is less understood, in particular on BBB integity mediated efflux transporters protein expressions. In this study we investigated the impact of repeated hypoxic stress on two efflux transporter proteins (Pgp and MRP-1) of an in vitro BBB model. For that, we have developed an in vitro BBB model composed of the immortalized mouse brain endothelial cells (bEnd.3) which was co-cultured in contact with the rat malignant glioma cells (C6). Hypoxic stress was induced by chemical agent: hydralazine (a mimetic agent of hypoxia pathway) during 2h and repeated during 24h or by physical hypoxia (2% O2). We assessed the functionality of Pgp and MRP-1 by measuring the transport of BCECF-AM, a fluorescent compound after repeated hypoxic stress. Then we investigated Pgp and MRP-1 expressions of our BBB model after hypoxic stress, by cell-ELISA method. We showed that endothelial cells increased their efflux transporter’s activity after repeated hypoxic stress. Indeed the level of extracellular BCECF (BCECF which was efflux by Pgp and MRP-1) was significantly increased after hypoxic stress (p<0.001). The concentration of extracellular BCECF varied from 4.43 ± 1.01 to 8.84 ± 0.47 µg/ml after the first exposition and to 11.13 ± 0.72 µg/ml after the third exposition. This increase of transport was associated to a significant increase of Pgp and MRP-1 expression (p<0.01). We observed that Pgp and MRP-1 expressions doubled between controls and cells exposed to repeated hypoxic stress (3.99 ± 1.14 versus 8.72 ± 0.27 µg/ml for Pgp, 9.21 ± 3.14 versus 19.97 ± 7.35 for MRP-1). No studies have specifically addressed the effect of repeated hypoxic stress on transporters, in brain endothelial cells. Ours results showed that brain microvascular endothelial cells had set up defence mechanism by increasing Pgp and MRP-1 activities. This increase of activity was associated to an increase of their expression. In conclusion endothelial cells of the BBB increased the expression of Pgp and MRP-1 to fight against the entry of potential cytotoxic compounds in the brain.