<p>Glioblastoma is the most prevalent and aggressive primary brain tumor in adults and current treatments result in only minimal improvements in survival. Studying the tumor microenvironment could lead to the development of more effective therapeutic alternatives. In this study, the inflammatory mechanisms involved in the interaction between glioma cells and microglia were explored using organotypic hippocampal cultures. Additionally, an in vivo glioblastoma model was used to confirm the involvement of microglia in glioblastoma malignancy. After 3 h of interaction between C6 glioma cells and organotypic cultures, there was a significant increase in the levels of the cytokines TNF-α, IL-1β, IL-6, and IL-10 in the medium, along with elevated Iba-1 immunoreactivity, indicating microglial activation. Microglia inhibition or depletion by treating organotypic cultures with minocycline or PLX3397 resulted in reduced levels of all the evaluated cytokines in the medium, confirming the role of microglia in the inflammatory microenvironment of glioblastoma. The interaction between glioblastoma cells and organotypic cultures led to increased levels of dedifferentiation markers CD133 and nestin in tumor cells, which were minimized after minocycline and PLX3397 treatment. However, no significant changes in cell migration or proliferation were observed in vitro after microglial inhibition or depletion. In vivo, minocycline treatment reduced the tumor volume and alleviated the histopathologic features of glioblastoma. These findings provide valuable insights into how microglia interact with tumors and healthy cells in the tumor microenvironment, driving neuroinflammation and tumor cell dedifferentiation. This understanding could pave the way for the development of innovative therapies for glioblastoma.</p> Graphical Abstract <p></p>