Staphylococcus aureus is one of the main bacterial species of clinical importance. Its virulence is considered multifactorial and is attributed to the combined action of a variety of molecular determinants including the virulence regulator SarA. Phosphorylation of SarA was observed to occur in vivo. From this finding, SarA was overproduced and purified to homogeneity. In an in vitro assay, it was found to be unable to autophosphorylate, but was effectively modified at threonine and serine residues by each of the two Ser/Thr kinases of S. aureus, Stk1 (PknB) and SA0077, respectively. In addition, phosphorylation of SarA was shown to modify its ability to bind DNA. Together, these data support the concept that protein phosphorylation directly participates, at the transcription level, in the control of bacterial pathogenicity.