Cancer cell metastasis is a major cause of death for breast cancer patients, and iron overload due to abnormal iron metabolism could promote proliferation and migration of breast cancer cells. The deferoxamine (DFO) is widely used, which inhibits the proliferation of tumor cells and decreases the iron content in estrogen receptor (ER)-positive human breast cancer cells but increases the iron content in triple-negative breast cancer (TNBC) cells. In addition, DFO could increase the iron content of mitochondria. However, the source of iron in mitochondria remains unclear. Iron metabolism and autophagy-related proteins in aggressive breast cancer MDA-MB-231 and nonaggressive breast cancer MCF-7 were examined by immunofluorescence, western blot and inductively coupled plasma-mass spectrometry. The possible regulatory mechanism of increased mitochondrial iron was explored by autophagy inhibitor. In this study, we found that iron content in mitochondria was due to the degradation of Ferritin by DFO-induced autophagy, which was essential for DFO-induced increase in cell epithelial mesenchymal transition and cell migration.Moreover, the increased level of mitochondrial calcium uniporter (MCU) and mitoferrin1(Mfrn1) might be involved in the DFO-induced increase of iron content in mitochondria. The study provides a research base for exploring new approaches to target iron metabolism for triple negative breast cancer in the clinic.