The standard therapy for glioblastoma comprises macroscopic complete resection and the adjuvant irradiation (IR). Often, the diffuse net-like infiltration of the brain parenchyma by highly migrating glioblastoma cells doesn't allow complete tumor resection and capture of all tumor cells by the IR target volume resulting in therapy failure. Migrating glioblastoma cells have to squeeze between very narrow interstitial spaces that reportedly requires local cell volume decrease mediated by Ca2+-activated BK K+ channels and Ca2+/calmodulin dependent kinase II (CaMKII)-stimulated ClC-3 Cl--channels. Importantly, ionizing radiation (IR) has been demonstrated to further stimulate migration of glioblastoma cells. Therefore, we tested for an IR-induced increase in BK K+ channel, Cl- channel, and CaMKII activities and their effects on cell migration. T98G glioblastoma cells were X-ray-irradiated with 0–2 Gy, migration was assessed by trans-well migration assay, BK K+- and Cl- channel activity by patch-clamp recording, cytosolic free Ca2+ concentration ([Ca2+]i) by Fura-2 Ca2+ imaging, and CaMKII activity by immunoblotting. As a result, IR modified [Ca2+]i and activated CaMKII which was sensitive to the BK channel inhibitor paxilline. In addition, IR increased the open probabilities of BK K+ channels and outwardly rectifying Cl- channels. The activity of the Cl- channels was lowered by paxilline pretreatment. Finally, IR stimulated the migration of T98G cells which was sensitive to paxilline and the CaMKII inhibitor KN-93. We conclude that enhanced BK channel activity and resulting activation of CaMKII and anion channels augments migration of irradiated glioblastoma cells.