An in vitro model of the blood-brain barrier (BBB), consisting of porcine brain-derived microvascular endothelial cells (PBMEC), was used to evaluate the effect of astrocytes in the BBB disruption during hypoxia. Hypoxia-induced hyperpermeability was decreased significantly in a coculture model of astroglia cells, either astrocytes or C6 glioma cells, with PBMEC and, to the same extent, when glia cell-conditioned medium was used. Corresponding to effects on hypoxia-induced hyperpermeability, astrocyte- and C6 cell-conditioned medium diminished hypoxia-induced vascular endothelial growth factor (VEGF) mRNA and protein expression, which recently was shown to be responsible for hypoxia-induced permeability changes in vitro. The effect on hypoxia-induced hyperpermeability and VEGF expression was specific for astroglia cells because conditioned medium from bovine smooth muscle cells (BSMC) did not show any effect. Immunocytochemistry revealed that 24 h of hypoxia disrupted the continuity of the tight junction protein, zonula occludens-1 (ZO-1), which lines the cytoplasmic face of intact tight junctions. These changes were prevented when hypoxia was performed in glia cell-conditioned medium. Results suggest that astrocytes protect the BBB from hypoxia-induced paracellular permeability changes by decreasing hypoxia-induced VEGF expression in microvascular endothelial cells.