Signaling through Toll-like receptors (TLRs), crucial molecules in the induction of host defense responses, requires adaptor proteins that contain a Toll/interleukin-1 receptor (TIR) domain. The pathogen <i>Staphylococcus aureus</i> produces several innate immune-evasion molecules that interfere with the host's innate immune response. A database search analysis suggested the presence of a gene encoding a homologue of the human TIR domain in <i>S. aureus</i> MSSA476 which was named staphylococcal TIR domain protein (TirS). Ectopic expression of TirS in human embryonic kidney, macrophage and keratinocyte cell lines interfered with signaling through TLR2, including MyD88 and TIRAP, NF-κB and/or mitogen-activated protein kinase pathways. Moreover, the presence of TirS reduced the levels of cytokines MCP-1 and G-CSF secreted in response to<i> S. aureus</i>. The effects on NF-κB pathway were confirmed using <i>S. aureus</i> MSSA476 wild type, an isogenic mutant MSSA476&#x0394;<i>tirS</i>, and complemented MSSA476&#x0394;<i>tirS +</i>pTirS in a Transwell system where bacteria and host cells were physically separated. Finally, in a systematic mouse infection model, TirS promoted bacterial accumulation in several organs 4 days postinfection. The results of this study reveal a new <i>S. aureus</i> virulence factor that can interfere with PAMP-induced innate immune signaling in vitro and bacterial survival in vivo.