Miro1 is a mitochondrial outer membrane protein that regulates mitochondrial and peroxisome trafficking, endoplasmic reticulum (ER) association, and mitophagy. Prior studies suggest a role for Miro1 in cell migration and proliferation in both normal and tumor cells. High Miro1 expression is associated with poor overall survival in breast cancer patients. To investigate the role of Miro1 in breast cancer tumorigenesis and metastasis, we established stable Miro1 knockdown (KD) in MDA-MB-231 human triple-negative breast cancer cells using shRNA. Miro1 KD significantly impaired cell proliferation, migration, and invasion in vitro. When implanted into the mammary fat pad of SCID mice, MDA-MB-231 cells formed tumors, whereas Miro1 KD cells showed markedly reduced tumorigenesis. Additionally, we generated a novel transgenic mouse model with inducible, tissue-specific Miro1 deletion in mammary epithelial cells, alongside polyomavirus middle T-antigen (PyVMT) oncogene activation. In this model, wild-type (WT) mice formed tumors at all mammary gland sites, with frequent lung metastases. However, Miro1-deficient mice failed to develop tumors, while heterozygous mice exhibited reduced tumor growth and metastasis. Additionally, these findings identify Miro1 as a key regulator of breast cancer onset and metastatic potential, positioning it as a potential biomarker and therapeutic target.