Connexins (Cxs), the gap junction proteins, have been found to be downregulated in many types of cancers including gliomas. By restoring gap junctional communication in cancer cell models, the neoplastic phenotype can be reversed, suggesting Cxs are tumor suppressors. Pannexin2 (Panx2) is a member of the novel gap junction protein family, Panxs, and it has been proposed as a brain-specific protein. Recently, gene array analysis showed an overall reduction of Panx2 in gliomas, and a direct correlation was observed between Panx2 expression and post-diagnosis survival in patients. In this study, we explored the potential inverse correlation between Panx2 and glioma oncogenicity. A decrease or absence of Panx2 expression in a panel of human glioma cell lines was found, whereas an appreciable amount of Panx2 was detected in both human brain and astrocytes. Stable Panx2 expression revealed a flattened morphology and increased cell-cell contacts in rat C6 glioma cells similar to Panx1. However, in contrast to Panx1 and Panx3, Panx2 was predominately detected in the cytoplasm in vesicle-like patterns but not at the plasma membrane. Coexpression of Panx2 and Panx1 did not show colocalization of both Panxs. Strikingly, restoration of Panx2 expression significantly reduced in vitro oncogenicity parameters, including monolayer saturation density and anchorage-independent growth, as well as in vivo tumor growth. This study suggests a role of aberrant Panx2 expression during gliomagenesis, and that Panx2 independently functions as a negative growth regulator without Panx1.