Abstract BACKGROUND Glioblastoma (GBM), the most lethal primary brain tumor, has a dismal prognosis, with a median survival of only 8 months and a 5-year survival rate of only 6.9%. Despite extensive research, the mechanisms underlying GBM resistance to conventional therapies remain unclear. Although the tolerance of GBM cells to chemoradiation is well established, the impact of surgical resection on residual tumor cells and their subsequent therapeutic response has been largely unexplored. This study aimed to elucidate the cellular plasticity changes induced by surgery in GBM cells and their potential implications for treatment resistance. MATERIAL AND METHODS A new mouse model for surgical resection of GBM guided by stereotaxic fluorescence was developed to mimic the surgical measures taken in patients. Syngeneic CT2A-NS (neurospheres), GL261-NS GBM cell lines, and patient-derived P3 cells were orthotopically implanted in immunocompetent C57Bl6J mice or immunocompromised nude mice. Pre- and post-surgical tumor tissues were studied using a multilevel strategy comprising time-resolved RNA-seq, immunohistochemistry, and intravital multiphoton microscopy. RESULTS Immunohistochemical analysis revealed that surgery-induced ischemia leading to a loss of perfused blood vessels and subsequent areas of necrosis close or far from the surgical cavity. Additionally, the remaining tumor blood vessels exhibited increased permeability, further exacerbating the tumor vascular dysfunction, as evidenced by intravital microscopy. This hypoxic/ischemic microenvironment significantly impacted residual GBM cells, driving a pronounced proneural-to-mesenchymal transition (PMT) and activating hypoxic pathways. Moreover, to investigate the impact of surgery-induced cell plasticity on therapy response, we investigated the response to irradiation in GBM cells collected either from untreated mice or 24 hours after neurosurgery. Post-surgical GBM cells exhibited significantly greater resistance to sublethal irradiation, associated with enhanced DNA damage repair capacity, increased self-renewal capability and tumorigenic potential. CONCLUSION Although surgery is an essential treatment for increasing the chances of survival of patients with GBM, it has a significant impact on GBM cell fate, inducing an ischemic microenvironment that drives PMT in GBM cells and may hinder a subsequent response to subsequent chemoradiotherapy.