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OBJECTIVE: To explore the possible mechanism under enhanced migration and proliferation of endothelial cells by glioma stem cells (GSCs).METHOD: Tumor spheres derived frommouse glioma cell line GL261,and brain micro‐vessel endothelial cell line b.END3 were used in this research.Transwell co‐culture system,RNAi experiment,Real‐time PCR,Western‐Blot,cck‐8 proliferation assay,Transwell migration assay and wound healing assay were used in this study to determine the migration and proliferation ability and related gene expression of Hedgehog pathway in b.END3 cells..RESULT: 1) Migration and proliferation abilities of endothelial cells were enhanced after co‐culture with GSCs.2) Related gene expression of Hedgehog pathway in endothelial cells was altered when co‐cultured with GSCs.Expression of Gli1 was up‐regulated.3) After knock‐down of Smo in endothelial cells,Hedgehog pathway was inhibited.Migration and proliferation abilities of endothelial cells,were both inhibited.GSCs have little effect on enhancing these behaviors in Smo‐knockdown endothelial cells.4) Further investigation revealed that Shh expression in endothelial cells and in supernatant of co‐culture system were elevated.CONCLUSION: GSCs can regulate migration and proliferation abilities of endothelial cells in vitro,probably related to activation of Hedgehog pathway,and increased expression of Shh in endothelial cells.