Previously, we demonstrated that high levels of mtDNA were functionally associated with “stemness” and aggressive phenotypic behaviours in human breast cancer cells, including spontaneous metastasis. More specifically, we showed that treatment with Alovudine induced mtDNA-depletion in MDA-MB-231 cells and prevented their ability to form colonies in vitro and metastasize in vivo. To better understand the underlying mechanism(s) and potentially develop a panel of protein biomarkers for metastasis, Alovudine- treated MDA-MB-231 cells were subjected to proteomics analysis. For comparison purposes, mtDNA-depletedrho(0) cells (MDA-MB-231 and MCF-7) were generated and also subjected to proteomics analysis. Intersection of these three distinct data sets revealed that a small number of proteins were commonly down-regulated in mtDNA-depleted rho(0) cells and Alovudine-treated cells. Remarkably, many of these nuclear-encoded mitochondrial genes (>20) were found to be genomically-amplified in human breast cancer metastatic samples. Therefore, the action of a single drug, namely Alovudine, was sufficient to effectively suppress the expression of a large number mitochondrial genes clinically associated with i) gene amplification and ii) cancer cell metastasis, in human breast cancer patients. These findings may have important clinical implications for the development of new therapeutics targeting cancer cell metastasis.