Microglia are the main immune effector cells in the central nervous system (CNS) and contribute to a wide spectrum of neuropathological conditions through exacerbated activation. Microglial inflammatory responses may be conditioned by prior exposures to noxious stimuli, such as increased levels of extracellular adenosine and ATP. These levels are characteristic of brain insults like epileptic seizures and could potentially determine subsequent responses through epigenetic regulation. In this study, we investigated DNA methylation and expression changes in microglia-like cells differentiated from monocytes following ATP-mediated preconditioning. First, during microglia differentiation, we mainly observed DNA demethylation in genomic sequences enriched in binding motifs of microglia lineage transcription factors like PU.1, consistent with the relevance of this factor in in vivo microglia. TLR-mediated activation, after a first exposure to ATP, promoted exacerbated pro-inflammatory activation in comparison to cells not pre-exposed to ATP. These changes were accompanied by DNA methylation and transcriptional reprogramming of immune-related pathways, neurotransmitter activity and nucleotide phosphorylation. Finally, the epigenetic and transcriptional reprogramming associated with ATP-mediated preconditioning results in profiles found in microglia subsets linked to epilepsy. Purine-driven microglia immune pre-conditioning associates with epigenetic and transcriptional changes that might contribute to altered functions of microglia during seizure development and progression, particularly associated with neuroinflammation.