Glioblastoma multiforme (GBM) is a highly therapy-resistant primary brain tumor, especially due to the presence of Glioblastoma Stem Cells (GSCs). In fact, GBM treatment imposes a combination of brain surgery, radiotherapy and chemotherapy with Temozolomide (TMZ) but still fails to completely abolish the tumor. Given this, exploring differentiation induction in GSCs emerges as a promising strategy for GBM treatment. Accordingly, we aimed to investigate the mechanisms that regulate stemness maintenance and differentiation in GBM using Whole-Cell Patch Clamp, Western Blot, Immunofluorescence and the FUCCI system.We explored an innovative approach by exploiting the observation that GSCs exhibit a distinctly depolarized resting membrane potential (RMP), reside in G0 phase and evade therapies targeting proliferative cells in G1/S phase, like TMZ.Our study reveals that Voltage-Gated Sodium Channels (Nav) expression in GBM cells is associated with reduced survival and increased stemness markers expression. The pharmacological inhibition of Nav substantially reduced the self-renewal and multipotency of GSCs, concomitantly enhancing their degree of differentiation.Additionally, the channel's blockage resulted in a significant transition of GBM cells from G0 phase to G1 and G1/S phase, enhancing their susceptibility to TMZ, and simultaneously increased their degree of differentiation through the MAPK/ERK signaling pathway activation. Taken together, these evidences position Nav as a promising prognostic biomarker and therapeutic target for GBM patients, particularly in conjunction with Temozolomide. Targeting Nav channels not only enhances the effectiveness of current therapies but also offers insights into differentiation induction, potentially overcoming the challenges posed by therapy-resistant GSCs in GBM patients.