The coordination of complex tumor processes requires cells to rapidly modify their phenotypes using direct cell-cell communication through gap junction channels composed of connexins. Previous reports suggest that gap junctions are tumor suppressors based on connexin 43 (Cx43), but this hypothesis fails to consider the differences in connexin-mediated intercellular communication rate and ion selectivity that drive gap junction diversity. Using patient-derived specimens, we screened connexin proteins and found that glioblastoma cancer stem cells (CSCs) expressed Cx46, while Cx43 was predominantly expressed in non-CSCs. Targeting Cx46 compromised CSC proliferation, self-renewal, and tumor initiation. Consistent with the divergent physiological nature of intercellular communication and ion selectivity between Cx46 and Cx43, CSCs with higher Cx46 had an elevated intercellular communication rate and were more depolarized than non-CSCs. The difference in connexin subtype was responsible for these phenotypic differences; Cx46 knockdown reduced the communication rate of CSCs, and Cx43 knockdown increased the depolarization of non-CSCs. The differences between the two connexins were reflected in GBM patient survival: Cx46 expression correlated with poor prognosis, while Cx43 expression was not informative. Ongoing studies are identifying differentially transported signaling molecules that are responsible for CSC maintenance based on connexin subunits. As clinically relevant gap junction inhibitors including 1-Octanol are being tested for other neurological disorders (essential tremor), we evaluated if these inhibitors were effective in glioblastoma. We confirmed that CSCs possessed functional gap junctions and that inhibitors reduced communication. These inhibitors potently inhibited proliferation and self-renewal of CSCs compared with non-CSCs and neural progenitor cells. In established xenograft tumors, gap junction inhibitors suppressed tumor growth and had an additive effect when combined with Temozolomide. Taken together, our data demonstrate a pro-tumorigenic role of gap junctions that is dependent on connexin subunit expression and provide a rationale for gap junction targeting in glioblastoma.