Leptomeningeal metastasis (LM) is a devastating complication of advanced non-small cell lung cancer (NSCLC). Its diagnosis and monitoring can be challenging. Recently, extracellular vesicle (EV) miRNAs have become a new noninvasive diagnostic biomarker. The purpose of this study was to examine the clinical value and role of EV miRNAs in NSCLC-LM. Next-generation sequencing analysis revealed that miRNAs with differential expression of EVs in sera of patients with NSCLC with LM and non-LM were detected to identify biological markers for the diagnosis of LM. Cellular and in vivo experiments were conducted to explore the pathogenesis of EV miRNA promoting LM in NSCLC. In the present study, we first demonstrated that the serum level of EV-associated miR-374a-5p in patients with LM of lung cancer was much higher than that in patients without LM and was correlated with the survival time of patients with LM. Further studies showed that EV miR-374a-5p efficiently destroys tight junctions and the integrity of the cerebral microvascular endothelial cell barrier, resulting in increased blood-brain barrier permeability. Mechanistically, miR-374a-5p regulates the distribution of ZO1 and occludin in endothelial cells by targeting gamma-adducin, increasing vascular permeability and promoting LM.Implications: These results suggest that serum NSCLC-derived EV miR-374a-5p is involved in premetastatic niche formation by regulating the permeability of the blood-brain barrier to promote NSCLC-LM and can be used as a blood biomarker for the diagnosis and prognosis of NSCLC-LM.