Experimental evidence from human patients and animal models of diabetes has demonstrated that hyperglycemia increases blood-brain barrier (BBB) permeability, which is associated with increased risk of neurological dysfunction. However, the mechanism underlying high glucose-induced BBB disruption is not understood. Here we investigated the role of hypoxia-inducible factor-1 (HIF-1) in high glucose-induced endothelial permeability in vitro using mouse brain microvascular endothelial cells (b.End3). Our results demonstrated that high glucose (30 mM) upregulated the protein level of HIF-1α, the regulatable subunit of HIF-1, and increased the transcriptional activity of HIF-1 in the endothelial cells. At the same time, high glucose increased the paracellular permeability associated with diminished expression and disrupted continuity of tight junction proteins occludin and zona occludens protein-1 (ZO-1) of the endothelial cells. Upregulating HIF-1 activity by cobalt chloride increased the paracellular permeability of the endothelial cells exposed to normal glucose (5.5 mM). In contrast, downregulating HIF-1 activity by HIF-1α inhibitors and HIF-1α specific siRNA ameliorated the increased paracellular permeability and the alterations of distribution pattern of occludin and ZO-1 induced by high glucose. In addition, high glucose increased expression of vascular endothelial growth factor (VEGF), a downstream gene of HIF-1. Inhibiting VEGF improved the expression pattern of occludin and ZO-1, and attenuated the endothelial leakage. Furthermore, key results were confirmed in human brain microvascular endothelial cells. These results strongly indicate that HIF-1 plays an important role in high glucose-induced BBB dysfunction. The results will help us understand the molecular mechanisms involved in hyperglycemia-induced BBB dysfunction and neurological outcomes.