Staphylococcus aureus (S. aureus) is a leading cause of Periprosthetic joint infection (PJI), a severe complication after joint arthroplasty. Immunosuppression is a major factor contributing to the infection chronicity of S. aureus PJI, posing significant treatment challenges. This study investigates the relationship between the immunosuppressive biofilm milieu and S. aureus PJI outcomes in both discovery and validation cohorts. This scRNA-seq analysis of synovium from PJI patients reveals an expansion and heightened activity of monocyte-related myeloid-derived suppressor cells (M-MDSCs) and regulatory T cells (Treg). Importantly, CXCL16 is significantly upregulated in M-MDSCs, with its corresponding CXCR6 receptor also elevated on Treg. M-MDSCs recruit Treg and enhance its activity via CXCL16-CXCR6 interactions, while Treg secretes TGF-β, inducing M-MDSCs proliferation and immunosuppressive activity. Interfering with this cross-talk in vivo using Treg-specific CXCR6 knockout PJI mouse model reduces M-MDSCs/Treg-mediated immunosuppression and alleviates bacterial burden. Immunohistochemistry and recurrence analysis show that PJI patients with CXCR6