S. aureus can grow in high salinity condition by a salt-tolerance mechanism. We have shown that S. aureus accumulated a large amount of proline in cytoplasm for the osmoregulation in high osmotic condition. In this study, we tried to elucidate the dynamics of proline by altering the salt concentration and supplementing proline in semisynthetic or synthetic media. S. aureus required only 20μM of proline as an essential amino acid under the normal condition, but 700μM to accumulate proline as an osmoregulator under the 10% NaCl condition. When the supplementary proline was not enough, S. aureus accumulated glutamine to compensate the osmoregulation by proline.