The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin 43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression. Display omitted •Gap junction targeting potently inhibits GBM growth•Gap junctions have a pro-tumorigenic role that depends on connexin expression•CSCs express Cx46, which is required for self-renewal•Connexin expression dictates intercellular communication and membrane potential Hitomi et al. demonstrate that glioblastoma cancer stem cells possess functional gap junctions that can be targeted to attenuate self-renewal and tumor growth. The authors also identify connexin 46 as a novel glioblastoma cancer stem cell regulator that is required for stem cell maintenance.