Cancer stem cells (CSCs) possess a greater invasiveness than their differentiated descendants with unknown mechanism. This study aimed to invastigate the mechanism of glioma stem cells (GSCs) participating in angiogenesis and invasion of maligmant glioma. We developed four novel methods for enrichment and isolation of GSCs from human glioblastoma cell lines and primary human glioma tissues, and studied the mechanismĀ of their self-renewal, multipotency, tumorigenicity, drug resistance, angigenesis and invasion. Techniques of cell sorting, immunostaining, laser cofocal and electron microscopy, chemotaxis, miRNA microarray, DNA methylation and siRNA, etc. were used in this study. We found that: (1) Activation of either CXCR4 or FPR preferentially expressed on GSCs increased calcium mobilization and the production of pro-angiogenic factors VEGF and CXCL8. Tumor xenografts in SCID mice derived from GSCs cells grew faster with more abundant microvessels. (2) GSCs showed reduced gap junctional intercellular communication (GJIC). CSCs expressed markedly reduced connexins, Cx43 in particular, which are key components of gap junction. We also observed hypermethylation in the promoter of gap junction protein Ī±1 (GJA1), which encodes Cx43) gene promoter and increased expression of microRNAs targeting GJA1 transcripts in GSCs. Reconstitution of Cx43 partially restored GJIC between GSCs and decreased their capacity of self-renewal, invasiveness, angiogenesis and tumorigenicity. This was associated with upregulation of E-cadherin and inactivation of SDF-1alpha/CXCR4 signaling pathway. Our results indicate that (1) GSCs contribute to tumor angiogenesis through, at least in part, the activation of CXCL12/CXCR4 and/or fMLF/FPR axes, which might be potential targets for glioma therapy, and (2) Cx43 is essential for normal GJIC and is capable of reversing the malignant phenotype of GSCs.(This study was supported by grants from the National Basic Research Program (973 Program, No.2006CB708503) and National Natural Science Foundation of China (NSFC, No. 30725035).