The activation of ion channels is critical for cell migration, including glioblastoma tumor cell movement in the brain parenchyma, that makes this tumor the most aggressive form of human brain tumors. In this context, we describe the contribution of the intermediate-conductance calcium-activated K (KCa3.1) channel activity in the chemotactic response to CXCL12, a chemokine whose expression in glioblastoma has been correlated with its invasive potential. In human glioblastoma cell lines we show that CXCL12 induces calcium mobilization, essential for CXCL12-induced chemotaxis, and activation and functional upregulation of the KCa3.1 channel. Blocking the KCa3.1 channel completely abolished CXCL12-induced glioblastoma cell migration. Further, we found that CXCL12 application to glioblastoma cells induces the activation of the ERK1/2 pathway which is only partially implicated in the modulation of CXCL12-induced glioblastoma cell movement, while the PI3-K pathway is not involved. These findings suggest that modulating the activity of KCa3.1 channel might represent a potential strategy to reduce glioblastoma invasiveness.