Abstract: Background　The intercellular interactions between human oligodendrocyte precursor cells (hOPCs) and human cerebral microvascular endothelial cells (hCMECs) participate in regulating the pathological progression of various central nervous system disorders, including white matter injury in premature infants and ischemic stroke, thereby playing a pivotal role in the onset and advancement of these diseases.Objective　This study investigates the interactions between hOPCs and hCMECs in an in vitro co-culture system and examines their roles in the treatment of relevant diseases.Methods　An in vitro co-culture model was built using Transwell cell culture chamber together with a co-culture medium that had been optimized in-house. hOPCs or human cerebral microvascular endothelial cell line D3(hCMEC/D3) maintained in co-culture were designated as the co-culture group, whereas their counterparts grown in monoculture served as the control group. Proliferation was measured by EdU incorporation, the migratory capacity of hOPCs by the Transwell assay, and the angiogenic capacity of hCMEC/D3 by the in vitro tube formation assay. RT-qPCR together with Western blot were applied to profile tight junction molecules of hCMEC/D3; RNA-seq was carried out to map transcriptome changes elicited in hCMEC/D3 by hOPCs; and ELISA quantified immunomodulatory cytokines of hCMEC/D3. Results　EdU labeling and Transwell migration assays revealed that hOPCs kept in co-culture proliferated more actively (P＜0.001) and migrated further (P＜0.01) than their monocultured counterparts. Consistent with this, hCMEC/D3 co-cultured with hOPCs showed enhanced proliferation (P＜0.05) and formed more extensive tube-like networks (P＜0.01) than controls. At the transcriptomic level, the presence of hOPCs drove upregulation of transcripts linked to proliferation, angiogenesis, tight junction, and a subset of immunomodulatory factors in hCMEC/D3 (P＜0.05, |log ₂ Fold Change| ≥1). RT-qPCR revealed that the mRNA expression level of CLDN5 in hCMEC/D3 was significantly higher in the co-culture group than in the control group (P ＜ 0.0001), while ZO-1 and OCLN showed an upward trend without statistically significant differences. Western blot confirmed the upregulation of CLDN5 at the protein level within the experimental group (P＜0.05). ELISA results showed that IL-6 and CXCL10 concentrations in the culture supernatants were likewise higher under co-culture conditions (P＜0.001).Conclusion　hCMEC/D3 and hOPCs engage in reciprocal crosstalk that jointly promotes proliferation, migration, and angiogenesis, while also raising the expression of tight junction components and immunomodulatory mediators—findings that may represent a promising therapeutic avenue for targeting neurovascular dysfunction-related disorders of the central nervous system and provides new experimental basis for cell therapy targeting central nervous system diseases.