The blood–brain barrier (BBB), consisting of specialized endothelial cells surrounded by astrocytes and pericytes, plays a crucial role in brain homeostasis. Many cerebrovascular diseases are associated with BBB breakdown and oxygen (O2) deprivation constitutes a critical factor that onsets its disruption. We investigated the impact of astrocytes and pericytes on brain endothelial cell permeability and survival during different degrees of O2 deprivation. Prolonged exposure to 1% O2 caused barrier breakdown and exposure to 0.1% O2 dramatically accelerated disruption and induced cell death, mediated at least in part via caspase-3 activation. Reoxygenation allowed only cells exposed to 1% O2 to re-establish barrier function. Notably co-culture with astrocytes and pericytes substantially enhanced barrier function under normoxic conditions, and produced differential responses during O2 deprivation. At 1% O2 astrocytes partially maintained barrier integrity whereas pericytes accelerated its disruption in the short-term, having positive effects only after prolonged exposure. Unexpectedly, at 0.1% O2 pericytes were more effective than astrocytes in preserving barrier function although the protection afforded by both cells involved inhibition of caspase-3 pathways. Furthermore, cell-specific regulation of auto- and paracrine VEGF signaling pathways were also in part responsible for the differential modulation of barrier function. Our data suggests that cellular cross-talk within the neurovascular unit is crucial for preservation of barrier integrity and that pericytes, not astrocytes, play a significant role during severe and prolonged O2 deprivation. J. Cell. Physiol. 218: 612–622, 2009. © 2008 Wiley-Liss, Inc.