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Glioblastoma (GBM) is the most frequent and malignant adult-type diffuse glioma (WHO grade IV) with a median patient survival of less than 15 months despite multimodal treatment. The poor prognosis of GBM, among others, is associated with a tumor-supporting microenvironment characterized by a rather anti-inflammatory state, where the tumor cells have been shown to hijack microglial gene expression profiles. These so-called tumor-associated microglia cells (TAMs) repress normal immune functions, such as phagocytosis and pro-inflammatory cytokine secretion through transcriptional reprogramming, further underlying morphological changes. It is currently starting to emerge that the exceptional cellular plasticity required to orchestrate such microglial reactivity could originate from pre-imposed epigenetic modifications. We thus hypothesize that TAM reprogramming in GBM is controlled by epigenetic mechanisms, and therefore aim to understand if modifications in DNA methylation patterns can lead to pro-tumor transcriptomic and phenotypical changes.Reactive microglia are classically described as either ‘pro-inflammatory’ M1 or ‘anti-inflammatory’ M2 phenotypes, inducable by Lipopolysaccharide (LPS) and Interferon-gamma (IFNɣ) or Interleukin-4 (IL-4), respectively. In a GBM context, microglial cells have been mostly classified as M2, but exhibit heterogeneous features among the M1 to M2 activation spectrum. In this study, we exposed human and murine microglial cell lines to LPS, IFNɣ, IL-4, and GBM-conditioned medium (GBM-CM), followed by morphological assessment, transcript expression and secretome characterization. The onset of activation was followed in time series every 12h to interrogate epigenetic and transcriptomic changes together with protein expression and secretion. After exposure to GBM-CM, microglial cells exhibited a bipolar and more elongated phenotype. We observed significant variations in cytokine production together with a decrease in phagocytosis in LPS-treated microglia, whereas the secretome in GBM-CM was only slightly altered. We furthermore observed differentially methylated regions (DMRs) using the novel Infinium® Mouse Methylation BeadChip, suggesting a role of DNA methylation in microglial reprogramming.