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This study identified and verified the characteristic differentially expressed ferroptosis-related genes (CDEFRGs) in osteosarcoma (OS). We extracted ferroptosis-related genes (FRGs), identified differentially expressed FRGs (DEFRGs) in OS, and conducted correlation analysis between DEFRGs. Next, we conducted GO and KEGG analyses to explore the biological functions and pathways of DEFRGs. Furthermore, we used LASSO and SVM-RFE algorithms to screen CDEFRGs, and evaluated its accuracy in diagnosing OS through ROC curves. Then, we demonstrated the molecular function and pathway enrichment of CDEFRGs through GSEA analysis. In addition, we evaluated the differences in immune cell infiltration between OS and NC groups, as well as the correlation between CDEFRGs expressions and immune cell infiltrations. Finally, the expression of CDEFRGs was verified through qRT-PCR, western blotting, and immunohistochemistry experiments. We identified 51 DEFRGs and the expression relationship between them. GO and KEGG analysis revealed their key functions and important pathways. Based on four CDEFRGs (PEX3, CPEB1, NOX1, and ALOX5), we built the OS diagnostic model, and verified its accuracy. GSEA analysis further revealed the important functions and pathways of CDEFRGs. In addition, there were differences in immune cell infiltration between OS group and NC group, and CDEFRGs showed significant correlation with certain infiltrating immune cells. Finally, we validated the differential expression levels of four CDEFRGs through external experiments. This study has shed light on the molecular pathological mechanism of OS and has offered novel perspectives for the early diagnosis and immune-targeted therapy of OS patients. [ABSTRACT FROM AUTHOR]