DMT1-dependent endosome-mitochondria interactions regulate mitochondrial iron translocation and metastatic outgrowth. DMT1 (SLC11A2) is a transporter that facilitates iron translocation from endosomes to mitochondria, playing a critical role in mitochondrial iron homeostasis. In triple-negative breast cancer (TNBC) cells, such as MDA-MB-231, DMT1 is involved in transient "kiss-and-run" interactions between endosomes and mitochondria, which are essential for mitochondrial iron translocation. However, in luminal A breast cancer cells like T47D, DMT1 does not perform this function. DMT1 silencing in MDA-MB-231 cells leads to increased labile iron pool (LIP) levels, activation of PINK1/Parkin-dependent mitophagy, and impaired mitochondrial bioenergetics. These changes are not observed in T47D cells. Transcriptomic analysis reveals that DMT1 knockout has different effects in 2D and 3D culture conditions, suggesting that the cellular environment is crucial for DMT1 function. In vivo studies show that DMT1 silencing in TNBC cells promotes lung metastasis. DMT1 is localized at endosomes and the outer mitochondrial membrane (OMM), acting as a bridge between endosomes and mitochondria. DMT1 silencing reduces EE-mitochondria interactions, mitochondrial iron translocation, and increases LIP levels, which are associated with increased metastatic potential. DMT1 also regulates mitochondrial iron translocation and mitophagy through interactions with PMPCB, a peptidase that controls PINK1 turnover. DMT1 silencing in TNBC cells leads to a novel iron phenotype characterized by reduced EE-mitochondria interactions, increased LIP levels, and enhanced metastatic outgrowth. These findings highlight the importance of DMT1 in maintaining mitochondrial iron homeostasis and metastatic fitness in TNBC cells. DMT1 expression is significantly reduced in metastatic tissues compared to primary tumors, suggesting a role in metastatic progression. DMT1 silencing in TNBC cells increases lung metastasis in vivo, indicating that DMT1 is critical for controlling metastatic growth. Overall, DMT1 is essential for maintaining mitochondrial iron homeostasis and regulating metastatic capacity in TNBC cells.DMT1-dependent endosome-mitochondria interactions regulate mitochondrial iron translocation and metastatic outgrowth. DMT1 (SLC11A2) is a transporter that facilitates iron translocation from endosomes to mitochondria, playing a critical role in mitochondrial iron homeostasis. In triple-negative breast cancer (TNBC) cells, such as MDA-MB-231, DMT1 is involved in transient "kiss-and-run" interactions between endosomes and mitochondria, which are essential for mitochondrial iron translocation. However, in luminal A breast cancer cells like T47D, DMT1 does not perform this function. DMT1 silencing in MDA-MB-231 cells leads to increased labile iron pool (LIP) levels, activation of PINK1/Parkin-dependent mitophagy, and impaired mitochondrial bioenergetics. These changes are not observed in T47D cells. Transcriptomic analysis reveals that DMT1 knockout has different effects in 2D and 3D culture conditions, suggesting that the cellular environment is crucial for DMT1 function. In vivo studies show that DMT1 silencing in TNBC cells promotes lung metastasis. DMT1 is localized at endosomes and the outer mitochondrial membrane (OMM), acting as a bridge between endosomes and mitochondria. DMT1 silencing reduces EE-mitochondria interactions, mitochondrial iron translocation, and increases LIP levels, which are associated with increased metastatic potential. DMT1 also regulates mitochondrial iron translocation and mitophagy through interactions with PMPCB, a peptidase that controls PINK1 turnover. DMT1 silencing in TNBC cells leads to a novel iron phenotype characterized by reduced EE-mitochondria interactions, increased LIP levels, and enhanced metastatic outgrowth. These findings highlight the importance of DMT1 in maintaining mitochondrial iron homeostasis and metastatic fitness in TNBC cells. DMT1 expression is significantly reduced in metastatic tissues compared to primary tumors, suggesting a role in metastatic progression. DMT1 silencing in TNBC cells increases lung metastasis in vivo, indicating that DMT1 is critical for controlling metastatic growth. Overall, DMT1 is essential for maintaining mitochondrial iron homeostasis and regulating metastatic capacity in TNBC cells.