Epidermal growth factor receptor (EGFR) is overexpressed in most triple-negative breast cancer (TNBC) patients with poor prognosis; however, the therapeutic benefit of EGFR inhibitors (EGFRi) in breast cancer remains limited. In this study, we found poor response to EGFRi in TNBC was related to oxidative phosphorylation (OXPHOS) and breast cancer stem cells (BCSCs), and demonstrated that TDP43 (TAR DNA-binding protein 43) expression is positively correlated with non-response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). TDP43 knockdown significantly enhances EGFR-TKI sensitivity and decreases EGFR-TKI resistance. Mechanistically, TDP43, a DNA/RNA-binding protein predominantly localized to the nucleus, translocates to mitochondria upon EGFR-TKI stimulation. The increased mitochondrial localization promotes OXPHOS, thereby enriching BCSCs and contributing to EGFR-TKI resistance. Inhibiting TDP43 expression or using our newly identified TDP43 inhibitor, atovaquone, suppresses OXPHOS and reduces EGFR-TKI resistance. Overall, our research identified TDP43 as a key regulator of EGFR-TKI sensitivity and resistance, and offers new therapeutic targets and promising application perspectives in TNBC.