Staphylococcus aureus is an opportunistic pathogen that causes a range of devastating diseases including chronic osteomyelitis, which partially relies on the internalization and persistence of S. aureus in osteoblasts. The identification of the mechanisms of the osteoblast response to intracellular S. aureus is thus crucial to improve the knowledge of this infectious pathology. Since the signal from specifically infected bacteria-bearing cells is diluted and the results are confounded by bystander effects of uninfected cells, we developed a novel model of long-term infection. Using a flow cytometric approach we isolated only S. aureus -bearing cells from mixed populations that allows to identify signals specific to intracellular infection. Here we present an in-depth analysis of the effect of long-term S. aureus infection on the transcriptional program of human osteoblast-like cells. After RNA-seq and KEGG and Reactome pathway enrichment analysis, the remodeled transcriptomic profile of infected cells revealed exacerbated immune and inflammatory responses, as well as metabolic dysregulations that likely influence the intracellular life of bacteria. Numerous genes encoding epigenetic regulators were downregulated. The later included genes coding for components of chromatin-repressive complexes ( e.g. , NuRD, BAHD1 and PRC1) and epifactors involved in DNA methylation. Sets of genes encoding proteins of cell adhesion or neurotransmission were also deregulated. Our results suggest that intracellular S. aureus infection has a long-term impact on the genome and epigenome of host cells, which may exert patho-physiological dysfunctions additionally to the defense response during the infection process. Overall, these results not only improve our conceptual understanding of biological processes involved in the long-term S. aureus infections of osteoblast-like cells, but also provide an atlas of deregulated host genes and biological pathways and identify novel markers and potential candidates for prophylactic and therapeutic approaches.