We performed in vivo RNA-sequencing analysis of Staphylococcus aureus in infected mouse liver using the 2-step cell-crush method. We compared the transcriptome of S. aureus at 6, 24, and 48 h post-infection (h.p.i) in mice and in culture medium. Genes related to anaerobic respiration were highly upregulated at 24 and 48 h.p.i. The gene expression patterns of virulence factors differed depending on the type of toxin. For example, hemolysins, but not leukotoxins and serine proteases, were highly upregulated at 6 h.p.i. Gene expression of metal transporters, such as iron transporters, gradually increased at 24 and 48 h.p.i. We also analyzed the transcriptome of mouse liver infected with S. aureus. Hypoxia response genes were upregulated at 24 and 48 h.p.i., and immune response genes were upregulated from 6 h.p.i. These findings suggest that gene expression of S. aureus in the host changes in response to changes in the host environment, such as oxygenation status or immune system attacks during infection.