ANO1, a Ca2+-activated chloride channel, is highly expressed in glioblastoma cells and its surface expression is involved in their migration and invasion. However, the regulation of ANO1 surface expression in glioblastoma cells is largely unknown. In this study, we found that Ca2+/Calmodulin-dependent protein kinase II (CaMKII) beta specifically enhances the surface expression and channel activity of ANO1 in U251 glioblastoma cells. When KN-93, a CaMKII inhibitor, was used to treat U251 cells, the surface expression and channel activity of ANO1 were significantly reduced. Only CaMKII beta, among the four CaMKII isoforms, increased the surface expression and channel activity of ANO1 in a heterologous expression system. Additionally, gene silencing of CaMKII beta suppressed the surface expression and channel activity of ANO1 in U251 cells. Moreover, gene silencing of CaMKII beta or ANO1 prominently reduced the migration and invasion of U251 and U87 MG glioblastoma cells. We thus conclude that CaMKII beta plays a specific role in the surface expression of ANO1 and in the ANO1-mediated tumorigenic properties of glioblastoma cells, such as migration and invasion.