The blood-brain barrier (BBB) is a cellular barrier formed by specialized brain endothelial cells under the influence of astrocytes and pericytes. Among the several stress factors known to induce BBB breakdown, hypoxia is probably the most represented but also the least understood. Recent evidence of oxidative stress occurring during hypoxia/ischemia situation raises its possible contribution to barrier breakdown. In this study, we investigated the relevance of oxidative stress in hypoxia-induced barrier disruption. Prolonged hypoxic exposure induced radical oxygen species (ROS) formation and induced glutathione oxidation. Such effects were accentuated under extreme O(2) deprived environment. Pro-oxidant treatment significantly disrupted barrier function under normal conditions, whereas anti-oxidant treatment contributed to maintain better barrier function and cell survival in an O(2)-reduced environment. In addition, the endothelial response to oxidative stress appeared modulated by the presence of astrocytes and pericytes, thus explaining some of the beneficial contribution of these cells as previously described. Taken together, this study highlights the importance of oxidative stress signaling at the barrier. In addition, cells of the neurovascular compartment differentially modulate ROS levels and also regulate barrier function. Thus, use of radical scavengers may be useful to support barrier function following stroke injury.