Initially described as colonizing livestock, Staphylococcus aureus from the clonal complex 398 (CC398) is now the causative organism of severe infections in humans. Several studies suggest that prophages acquisition may explain these evolutions. The project’s aim was to identify the molecular mechanisms triggered by prophages that modulate bacterial virulence and host tropism of CC398 isolates. Such findings could contribute to identify specific markers of bacterial virulence and/or epidemiological evolution useful for infection control and therapy. By constructing “model strains” containing or not prophages, we investigated the effect of prophages acquisition on strains capability to adhere to human proteins and to invade human non-phagocytic cells, as well as to invade and persist in aortic vegetations in a model of endocarditis in rats. Comparison of the transcriptomes of a strains pair containing or not prophages, followed by directed mutagenesis allowed identifying a possible novel mechanism by which prophages modulate S. aureus virulence.