The blood-tumor barrier (BTB) constitutes an efficient organization of tight junctions that limits the delivery of chemotherapeutic drugs to brain tumor tissues and impacts the treatment of glioma. Long non-coding RNAs (lncRNAs) are non-protein coding RNAs regulating gene expression, some lncRNAs play a crucial role in BTB permeability. However, the function of lncRNAs in BTB permeability is still largely unclear. Here, we have identified lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), was remarkably up-regulated in glioma endothelial cells (GECs) obtained from an in vitro BTB model. Knockdown of NEAT1 impaired the integrity and increased the permeability of the BTB, accompanied by downregulation of expression of the tight junction proteins ZO-1, occludin and claudin-5 in GECs. Both bioinformatics data and results of luciferase reporter assays demonstrated that NEAT1 influenced BTB permeability by binding to miR-181d-5p. Knockdown of NEAT1 also down-regulated the expression of sex determining region Y-box protein 5 (SOX5), which was defined as a direct and functional downstream target of miR-181d-5p. SOX5 interacts with the promoter region of ZO-1, occludin and claudin-5 in GECs. In conclusion, knockdown of NEAT1 increased BTB permeability by binding to miR-181d-5p and then reducing tight junction protein expression by targeting SOX5. These results suggest an important role for NEAT1 in regulating BTB permeability and provide an additional strategy for treating glioma.