Staphylococcus aureus is a highly versatile pathogen that can infect the tissue of human by producing a large arsenal of virulence factors, which was tightly regulated by a complex regulatory network. Rot, sharing sequence similarity to SarA homologues, is a global regulator that regulates numerous virulence genes. However, the recognition model of Rot to the promoter regions of target genes and the putative regulation mechanism remain elusive. In this study, we report the 1.77 Å resolution X-ray crystal structure of Rot. The structure reveals that two Rot moleculars form a compact homodimer, each of which contains a typical helix-turn-helix module and a β-hairpin motif connecting by a flexible loop. The fluorescence polarization results indicate that Rot preferentially recognizes AT-rich dsDNA ≈30 base pair nucleotides, and the conserved positively charged residues on the winged-helix motif are vital for binding to the AT-rich dsDNA. Taken together, we propose an interaction model that the helix-turn-helix motifs of each monomer of Rot interact with the major grooves of target dsDNA and the winged motifs contact the minor grooves, which may be similar to that of SarA, SarR and SarS. In addition, the structure shows that Rot adopts a novel dimer interaction model, and perturbation of the dimer interface abolishes the transcription regulation activity of Rot by depriving the AT-rich dsDNA binding ability. Our data hints that Rot has folds similar to other SarA homologues, but the putative regulation mechanism may be different.