Osteomyelitis is a challenging disease in the field of bone infections, with its immune and molecular regulatory mechanisms still poorly understood. The aim of this study is to explore the value and potential mechanisms of cuproptosis-related genes (CRGs) in Staphylococcus aureus (S. aureus)-infected osteomyelitis from an immunological perspective.Initially, three transcriptomic datasets from public databases were integrated and analyzed, and consistent expression of CRGs in S. aureus-infected osteomyelitis was identified. Subsequently, immune infiltration analysis was performed, and M2 macrophage-related CRGs (M2R-CRGs) were further identified. Their potential molecular mechanisms were evaluated using Gene Set Variation Analysis (GSVA) and Gene Set Enrichment Analysis (GSEA). Finally, distinct osteomyelitis subtypes and diagnostic models based on characteristic M2R-CRGs were constructed.Through correlation analysis with immune cell infiltration, three characteristic M2R-CRGs (SLC31A1, DLD, and MTF1) were identified. Further analysis using unsupervised clustering and immune microenvironment analysis indicated that cluster 1 might activate pro-inflammatory responses, while cluster 2 was shown to exhibit anti-inflammatory effects in osteomyelitis. Compared to Cluster A, Cluster B demonstrated higher levels and a greater diversity of immune cell infiltrations in CRG-related molecular patterns, suggesting a potential anti-inflammatory role in osteomyelitis. A diagnostic model for S. aureus-infected osteomyelitis, based on the three M2R-CRGs, was constructed, exhibiting excellent diagnostic performance and validated with an independent dataset. Significant upregulation in mRNA and protein expression levels of the three M2R-CRGs was observed in rat models of S. aureus-infected osteomyelitis, aligning with bioinformatic results.The M2R-CRGs (SLC31A1, DLD, and MTF1) may be considered characteristic genes for early diagnosis and personalized immune therapy in patients with S. aureus-infected osteomyelitis.© 2024 Shi et al.