Using A/J mice, which are susceptible to Staphylococcus aureus, we sought to identify genetic determinants of susceptibility to S. aureus, and evaluate their function with regard to S. aureus infection. One QTL region on chromosome 11 containing 422 genes was found to be significantly associated with susceptibility to S. aureus infection. Of these 422 genes, whole genome transcription profiling identified five genes (Dcaf7, Dusp3, Fam134c, Psme3, and Slc4a1) that were significantly differentially expressed in a) S. aureus -infected susceptible (A/J) vs. resistant (C57BL/6J) mice and b) humans with S. aureus blood stream infection vs. healthy subjects. Three of these genes (Dcaf7, Dusp3, and Psme3) were down-regulated in susceptible vs. resistant mice at both pre- and post-infection time points by qPCR. siRNA-mediated knockdown of Dusp3 and Psme3 induced significant increases of cytokine production in S. aureus-challenged RAW264.7 macrophages and bone marrow derived macrophages (BMDMs) through enhancing NF-κB signaling activity. Similar increases in cytokine production and NF-κB activity were also seen in BMDMs from CSS11 (C57BL/6J background with chromosome 11 from A/J), but not C57BL/6J. These findings suggest that Dusp3 and Psme3 contribute to S. aureus infection susceptibility in A/J mice and play a role in human S. aureus infection.