Staphylococcus aureus (S. aureus) is one of the most important pathogens in humans and animals, multiply resistant strains are increasingly widespread, new agents are needed for the treatment of S. aureus. Rhein (RH), a natural plant product, has been reported to have potential antimicrobial activity against S. aureus, but the response mechanisms of S. aureus to RH are still poorly understood. RH showed good in vitro antibacterial activity against all 21 tested S. aureus strains in this experiment. We performed commercial Affymetrix GeneChips to determine the overall transcriptional response of S. aureus ATCC25923 triggered by the treatment of subinhibitory concentrations of RH at one time point 45 min. A total of 88 genes were identified to be differentially regulated by RH. Of these, 28 transporter genes were differentially regulated by RH; RH stress elevated the transcription of genes (srtB and isdABCDEFGI) encoding iron-regulated surface determinants system and genes (nrdIEF and nrdDG) involved in ribonucleotide reductase systems; but RH repressed genes (pflAB, nirBDR, narGH, ldh1, COL-SA0660, COL-SA2363 and COL-SA2386) responsible for anaerobic respiration and fermentation. To our knowledge, this genome-wide transcriptomics approach revealed first insights into the response of S. aureus to RH challenge.