S. aureus is a major etiological agent of ruminant mastitis worldwide. Chronicity of S. aureus mastitis depends on strains’ characteristics, and makes this infection difficult to cure and prone to recurrence. To identify bacterial factors involved in chronicity, Newbould 305, a strain that reproducibly induces mild and chronic mastitis in experimental infections was characterized in-depth. We employed genomic and proteomic approaches, combined to phenotypic characterization, to comprehensively analyze Newbould 305. Results were compared to S. aureus RF122, a strain representative of the major clone involved in bovine mastitis worldwide. Variations were found in the content of mobile genetic elements. Newbould 305 presents a gamut of genes encoding toxins, proteases and surface exposed proteins that reflect host-adaptation and invasion capacities. Especially, presence and characteristics of these surface exposed proteins were correlated with higher cytotoxicity, and higher adhesion and internalization capacities in Newbould 305 into bovine mammary epithelial cells. Our data are consistent with the role of invasion into host cells and host-adaptation features in the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to define the underlying mechanisms of mastitis chronicity.