<p>Osteosarcoma (OS) is a malignant tumor whose chemoresistance severely compromises therapeutic efficacy. This study aims to investigate the molecular mechanism by which TIMM23 mediates M2 polarization of macrophages through mitophagy and regulates TIMM23-PARGP1 fusion gene expression and chemoresistance in OS. Single-cell transcriptomic analysis revealed a strong interaction between macrophages and tumor cells. By integrating bulk RNA sequencing data and weighted gene co-expression network analysis (WGCNA) co-expression network analysis, we identified TIMM23 as a key gene associated with macrophage polarization. Using STAR-Fusion, we further detected the TIMM23-PARGP1 fusion gene, which was validated via fluorescence in situ hybridization (FISH). CRISPR/Cas9 was employed to generate TIMM23-knockout macrophages, and a lentiviral system was used to create TIMM23-overexpressing macrophages. Flow cytometry demonstrated that TIMM23 promotes M2 polarization, while confocal and transmission electron microscopy confirmed its role in regulating mitophagy in M2 macrophages. These findings indicate that TIMM23 promotes M2 polarization through mitophagy modulation. Next, a co-culture model of macrophages and OS cells was established. MTT, Cell Counting Kit-8 (CCK-8), EdU, Transwell, and TUNEL assays were performed to evaluate tumor cell behavior and chemosensitivity. The results showed that TIMM23-induced M2 polarization upregulated TIMM23-PARGP1 expression in OS cells, thereby enhancing their proliferation, migration, and invasion while inhibiting apoptosis and reducing the effectiveness of chemotherapeutic agents. In vivo experiments further confirmed the role of TIMM23 in promoting M2 polarization and fusion gene expression, leading to increased chemoresistance and tumor growth. In conclusion, TIMM23 enhances OS chemoresistance and tumor progression by promoting M2 macrophage polarization via mitophagy and upregulating TIMM23-PARGP1 fusion gene expression.</p><p></p>