Staphylococcus aureus can colonize and infect both humans and animals, but isolates from both hosts tend to belong to different lineages. Our recent finding of bovine-adapted S. aureus showing close genetic relationship to the human S. aureus clonal complex 8 (CC8) allowed us to examine the genetic basis of host adaptation in this particular CC. Using total chromosome microarrays, we compared the genetic makeup of 14 CC8 isolates obtained from cows suffering subclinical mastitis, with nine CC8 isolates from colonized or infected human patients, and nine S. aureus isolates belonging to typical bovine CCs. CC8 isolates were found to segregate in a unique group, different from the typical bovine CCs. Within this CC8 group, human and bovine isolates further segregated into three subgroups, among which two contained a mix of human and bovine isolates, and one contained only bovine isolates. This distribution into specific clusters and subclusters reflected major differences in the S. aureus content of mobile genetic elements (MGEs). Indeed, while the mixed human-bovine clusters carried commonly human-associated β-hemolysin converting prophages, the bovine-only isolates were devoid of such prophages but harbored an additional new non-mec staphylococcal cassette chromosome (SCC) unique to bovine CC8 isolates. This composite cassette carried a gene coding for a new LPXTG-surface protein sharing homologies with a protein found in the environmental bacterium Geobacillus thermoglucosidans. Thus, in contrast to human CC8 isolates, the bovine-only CC8 group was associated with the combined loss of β-hemolysin converting prophages and gain of a new SCC probably acquired in the animal environment. Remaining questions are whether the new LPXTG-protein plays a role in bovine colonization or infection, and whether the new SCC could further acquire antibiotic-resistance genes and carry them back to human.