Although little is known about the regulatory mechanisms underlying the pathogenesis of osteomyelitis caused by Staphylococcus aureus (S. aureus), hypoxia-inducible factor-1α (HIF-1α) and STIP1 homology and U-box containing protein 1 (STUB1) have been found to be up-regulated in both S. aureus infected MC3T3-E1 cells and in patients with osteomyelitis. HIF-1α directly targets STUB1 to induce its expression. In MC3T3-E1 cells infected with S. aureus, silencing HIF-1α and STUB1 and administering the hypoxia inhibitor IDF-11774 consistently increased the expression of OSX and RUNX2, as well as the levels of alizarin Red S and alkaline phosphatase activity. In a mouse model of osteomyelitis, S. aureus infection elevated HIF-1α expression and serum STUB1 levels. Interleukin (IL)-6, IL-1β, and C-reactive protein levels in serum were reduced after treatment with the hypoxia inhibitor IDF-11774. Following an infection with S. aureus, hypoxia was activated to cause STUB1 overexpression by directly targeting HIF-1α, ultimately causing osteomyelitis symptoms such as osteogenesis and mineralization defected and increased inflammation. This study presents a novel signaling cascade in the pathogenesis of osteomyelitis involving hypoxia/HIF-1α/STUB1. This signaling cascade may be a target for therapeutic interventions.