Staphylococcus aureus has become a serious concern in hospitals and community due to rapid adaptation to existing antimicrobial agents. Betulinaldehyde [3?-hydroxy-20(29)-lupen-28-al (BE)] belongs to pentacyclic triterpenoids that are based on a 30-carbon skeleton comprising four six-membered rings and one five-membered ring. In a preliminary study, BE exhibited antimicrobial activity against reference strains of methicillin-resistant and methicillin-sensitive S. aureus. However, the response mechanism of S. aureus to this compound is not known. In this study, the global gene expression patterns of both the reference strains in response to sub-inhibitory concentrations of BE were analyzed using DNA microarray to identify gene targets, particularly essential targets in novel pathways, i.e. not targeted by currently used antibiotics, or novel targets in existing pathways. The transcriptome analysis revealed repression of genes in the aminoacyl-tRNA synthetase and ribosome pathways in both the reference strains. Other pathways such as cell division, two-component systems, ABC transporters, fatty acid biosynthesis and peptidoglycan biosynthesis were affected only in the reference strain of methicillin-resistant S. aureus. The findings suggest that BE regulates multiple desirable targets which could be further explored in the development of therapeutic agents for the treatment of S. aureus infections.