For the first time, a genome-wide transcriptional analysis was performed to elucidate the cellular response of Staphylococcus aureus to triclosan. Our results indicate that the effects of triclosan are widespread on metabolism, affecting many vital cellular processes. Triclosan downregulated the transcription of genes involved in virulence factor and energy metabolism such as amino acid, carbohydrate, lipid transport, and metabolism, while multidrug resistance genes, coenzyme transport, and metabolism and transcription genes were upregulated. Furthermore, triclosan downregulated the transcription of genes encoding major lipid metabolism enzymes such as 3-hydroxyacyl-CoA dehydrogenase, acetyl-CoA acetyltransferase, acetyl-CoA synthetase, and acetyl-CoA carboxylase, which all play essential roles in S. aureus lipid metabolism. It is interesting to note that the expression of the enoyl-ACP reductase gene, fabI, was not changed after exposure of S. aureus with 0.05 mu M triclosan at 10 and 60 min in our study. This work also implies that triclosan may kill S. aureus by interfering with its ability to form cell membranes. Another important implication of our result is that S. aureus may generate resistance factors under triclosan stress.