Background <p>Glioblastoma multiforme (GBM) is a highly aggressive astrocytic glioma with a devastating survival rate of less than 7%. Despite treatment with surgical resection and chemoradiotherapy, a majority of GBM cases recur. The intricate tumor microenvironment and the elusive nature of its recurrence are still controversial. Herein, we explore the role of neuronal hyperstimulation in glioblastoma cell regrowth post-chemotherapy, focusing on cancer-neuron interactions.</p> Methods <p>A direct electrical stimulation system, validated by COMSOL Multiphysics simulation, was used to induce stimulation of neuronal networks through the formation of an extremely low frequency (ELF) electric field, and changes by excitability were tracked. The custom-designed co-culture system, enabling the sharing of paracrine signals in an independent microenvironment cultivation of neuronal networks and glioblastoma cell, was employed to investigate the effects of neuronal excitability on glioblastoma cell.</p> Results <p>Power-frequency electric fields are applied to hippocampal neuronal networks to elicit abnormal neuronal activity, evidenced by calcium influx and neurotransmitter release. While temozolomide effectively suppresses glioblastoma cell proliferation, their co-culture with stimulated neurons reignites cancer growth. Blocking glutamate release from neuron networks counter the effects of neuronal activity, highlighting the significance of paracrine signaling in glioblastoma cell proliferation and recurrence.</p> Conclusions <p>Our findings illuminate a pathway through which environmental factors contribute to GBM regrowth following chemotherapy and propose a potential therapeutic target, neuron-cancer communication, to prevent GBM recurrence.</p>