High-grade gliomas (HGGs) include astrocytoma and glioblastoma. They are characterized by aggressiveness, intra- and inter-tumor heterogeneity, and infiltrating ability, decreasing the efficacy of resection and therapies. Glioma invasiveness depends on the dynamic interaction between glial cells, mainly microglia, and astrocytes. Accordingly, extra-CNS metastasis remains a rare clinical entity. Astrocytic connexin43 (Cx43) is under microglia control. Cx43-mediated signaling profoundly impacts HGGs invasion and could be a potential target in the glia-HGGs communication. However, it needs further insights.We dissected tumor and peritumoral cortex from human surgical specimens to characterize the role of Cx43 in the neuroglial network with the HGGs. Acute and organotypic slices were prepared for functional, molecular, and morphological characterization. Human primary glioblastoma cells were tagged using lentiviral transduction and injected into organotypic cortex of peritumoral tissue. Glioblastoma cells were tracked until div7 to test the tumor progression as well as their response to Gap19, a selective blocker of the Cx43 hemichannels. Peritumoral cortex reacts to ictal-like microenvironment, and differs from tumor activity. Microglia and macrophages morphology changes among the tumor and peritumoral tissue. The CD44 glycoprotein, which participates in cell-cell interaction and migration, has a differential expression pattern. GFAP- and Cx43-expressing cells decrease in the tumor area. Implanted glioblastoma cells invade the peritumoral slices, overexpress Cx43, and undergo polarization after exposure to Gap19, implying a modification of the extracellular matrix and limited migration.These findings shed light on Cx43 as a key player in the smart, plastic crosstalk between glial cells and tumors, prompting further studies.