Despite the widespread use of chlorination for microbial control in aqueous environments, cellular response mechanisms of human pathogens, such as Staphylococcus aureus, against chlorination remain unknown. In this work, genome-wide transcriptional analysis was performed to elucidate cellular response of S. aureusto hypochlorous acid, an active antimicrobial product of chlorination in aqueous solution. Our results suggest that hypochlorous acid repressed transcription of genes involved in cell wall synthesis, membrane transport, protein synthesis, and primary metabolism, while amino acid synthesis genes were induced. Furthermore, hypochlorous acid induced transcription of genes encoding major virulence factors of S. aureus, such as exotoxins, hemolysins, leukocidins, coagulases, and surface adhesion proteins, which all play essential roles in staphylococcal virulence. This work implies that chlorination may stimulate production of virulence factors, which provides new insight into host-pathogen interactions and effects of chlorine application for microbial control.