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Background: Alterations in Mitochondrial DNA methylation (MTDM) exist in many tumors, but their role in breast cancer (BC) development remains unclear. Methods: We analyzed BC patient data by combining scRNA-seq and bulk sequencing. Weighted co-expression network analysis (WGCNA) of TCGA data identified mitochondrial DNA methylation (MTDM)-associated genes in BC. COX regression and LASSO regression were used to build prognostic models. The biological function of MTDM was assessed using various methods, such as signaling pathway enrichment analysis, copynumber karyotyping analysis, and quantitative analysis of the cell proliferation rate. We also evaluated MTDM-mediated alterations in the immune microenvironment using immune microenvironment, microsatellite instability, mutation, unsupervised clustering, malignant cell subtype differentiation, immune cell subtype differentiation, and cell-communication signature analyses. Finally, we performed cellular experiments to validate the role of the MTDM-associated prognostic gene NCAPD3 in BC. Results: In this study, MTDM-associated prognostic models divided BC patients into high/low MTDM groups in TCGA/GEO datasets. The difference in survival time between the two groups was statistically significant (P<0.001). We found that high MTDM status was positively correlated with tumor cell proliferation. We analyzed the immune microenvironment and found that low-MTDM group had higher immune checkpoint gene expression/immune cell infiltration, which could lead to potential benefits from immunotherapy. In contrast, the high MTDM group had higher proliferation rates and levels of CD8+T cell exhaustion, which may be related to the secretion of GDF15 by malignant breast epithelial cells with a high MTDM status. Cellular experiments validated the role of the MTDM-associated prognostic gene NCAPD3 (the gene most positively correlated with epithelial malignant cell proliferation in the model) in BC. Knockdown of NCAPD3 significantly reduced the activity and proliferation of MDA-MB-231 and BCAP-37 cells, and significantly reduced their migration ability of BCAP-37 cell line. Conclusion: This study presented a holistic evaluation of the multifaceted roles of MTDM in BC. The analysis of MTDM levels not only enables the prediction of response to immunotherapy but also serves as an accurate prognostic indicator for patients with BC. These insightful discoveries provide novel perspectives on tumor immunity and have the potentially to revolutionize the diagnosis and treatment of BC. [ABSTRACT FROM AUTHOR]