Mitochondria contain a 16-kb double stranded DNA genome encoding 13 proteins essential for respiration, but the mechanisms regulating transcription and their potential role in cancer remain elusive. Although methyl-CpG-binding domain (MBD) proteins are essential for nuclear transcription, their role in mitochondrial DNA (mtDNA) transcription is unknown. Here we report that the MBD2c splicing variant translocates into mitochondria to mediate mtDNA transcription and increase mitochondrial respiration in triple-negative breast cancer (TNBC) cells. In particular, MBD2c binds the noncoding region in mtDNA and interacts with SIRT3, which in turn deacetylates and activates TFAM, a primary mitochondrial transcription factor, leading to enhanced mtDNA transcription. Furthermore, MBD2c recovered the decreased mitochondrial gene expression caused by the DNA synthesis inhibitor cisplatin, preserving mitochondrial respiration and consequently enhancing drug resistance and proliferation in TNBC cells. These data collectively demonstrate that MBD2c positively regulates mtDNA transcription, thus connecting epigenetic regulation by deacetylation with cancer cell metabolism, suggesting druggable targets to overcome resistance.