Glioblastoma (GBM) is the most malignant form of primary brain tumor. It is characterized by the presence of highly invasive cancer cells infiltrating the brain by interacting with astrocytes and endothelial cells. During invasion, GBM cells significantly shrink their volume and extend lamellipodia by modulating their membrane conductance repertoire. However, the changes in compartment-specific ionic dynamics involved in this process are still not fully understood. To investigate the alterations in the GBM cells membrane conductance during lamellipodia formation and migration, a battery of complementary assays and methods were used: non-invasive perforated patch-clamp, wound-healing assay, time-lapse microscopy, and immunofluorescence reactions. By wound-healing assay, the migrating cells were recognized by digitiform structure, known as lamellipodia, oriented toward the empty area, and defined by us as “edge cells”. The results demonstrate that the Na+/Ca2+ exchanger (NCX), highly expressed in the lamellipodia, is functionally active during GBM cell migration, and is associated with the overexpression of the Large-conductance Ca2+-activated K+ (BK) and/or the specific glioma form (gBK) channels. Furthermore, NCX blockade impairs lamellipodia formation and maintenance affecting GBM cells migration. These data suggest the crucial role of NCX functional expression in GBM cells’ invasion, paving the way to consider the NCX a potential target for brain cancer treatment.This research was funded by: Italian Ministry of Health, Traiettoria 4 del Piano Operativo Salute (POS) linea di Azione 4.1 “Creazione di Hub delle Scienze della Vita” Project IMMUNOHUB, T4-CN-02