Glioblastoma multiforme (GBM) is the most common brain cancer in adults. GBM starts from a small fraction of poorly differentiated and aggressive cancer stem cells (CSCs) responsible for aberrant proliferation and invasion. Due to extreme tumor heterogeneity, actual therapies provide poor positive outcomes, and cancers usually recur. Therefore, alternative approaches, possibly targeting CSCs, are necessary against GBM. Among emerging therapies, high intensity ultra‐short pulsed electric fields (PEFs) are considered extremely promising and our previous results demonstrated the ability of a specific electric pulse protocol to selectively affect medulloblastoma CSCs preserving normal cells. Here, we tested the same exposure protocol to investigate the response of U87 GBM cells and U87‐derived neurospheres. By analyzing different in vitro biological endpoints and taking advantage of transcriptomic and bioinformatics analyses, we found that, independent of CSC content, PEF exposure affected cell proliferation and differentially regulated hypoxia, inflammation and P53/cell cycle checkpoints. PEF exposure also significantly reduced the ability to form new neurospheres and inhibited the invasion potential. Importantly, exclusively in U87 neurospheres, PEF exposure changed the expression of stem‐ness/differentiation genes. Our results confirm this physical stimulus as a promising treatment to destabilize GBM, opening up the possibility of developing effective PEF‐mediated therapies. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

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