Osteomyelitis is a serious bacterial infection of bone that is associated with progressive inflammatory tissue damage. This condition commonly affects the long bones of children along with the vertebrae and pelvis of adults. Staphylococcus aureus, the principal causative agent of osteomyelitis, can enter bone via the bloodstream or surrounding tissues following injury or surgery resulting in disease that is often refractory to therapies such as debridement or antibiotic treatment. Furthermore, the increasing incidence of infections associated with antibiotic resistant strains of S. aureus has compounded this problem such that new treatment strategies are needed urgently. Bone-forming osteoblasts can be infected by S. aureus and serve as an intracellular bacterial reservoir for chronic infection. However, this infection can be perceived via an array of microbial pattern recognition receptors that are known to be expressed by osteoblasts, triggering changes in their function. For example, these cells play a critical role in the control of the formation and activity of bone resorbing osteoclasts, and we have shown that they can exacerbate inflammation at sites of infection by producing an array of cytokines, chemokines, and growth factors. As such, infected osteoblasts play an important role in the abnormal bone formation and inflammatory damage associated with S. aureus infection. Type I interferons are best known for their antiviral effects, but it is becoming increasingly apparent that they can impact host susceptibility to a wide range of pathogens including S. aureus. Furthermore, two studies have suggested that osteoblasts might be capable of producing the type I interferon, IFN-β, either constitutively or following exposure to pattern recognition receptor agonists. In the present study, we have assessed the ability of primary murine osteoblasts to produce and respond to IFN-β following infection with S. aureus strain UAMS-1. The production of IFN-β and interferon stimulated genes were assessed by specific capture ELISAs and immunoblot analyses. IFN-β production was confirmed with the demonstration that S. aureus induces its rapid and robust release by osteoblasts in a dose-dependent manner. We have also shown increased protein expression of the interferon stimulated gene products IFIT1 and IFIT3 by infected OBs and demonstrated that this occurs secondary to the release of IFN-β by these cells. Finally, we have determined that exposure of S. aureus-infected osteoblasts to IFN-β markedly reduces the number of viable bacteria harbored by these cells. Together with studies performed previously by our laboratory, these findings indicate an ability of osteoblasts to respond to bacteria by producing IFN-β that can act in an autocrine and/or paracrine manner to elicit interferon stimulated gene expression and mitigate S. aureus infection.