Mitochondrial DNA (mtDNA) mutations can regulate tumor cell metastasis. This study used cybrid technology to transfer mtDNA between metastatic and non-metastatic mouse tumor cells, revealing that mtDNA mutations can enhance metastatic potential. Mutations in the ND6 gene, such as G13997A and 13885insC, cause respiratory complex I dysfunction and increased reactive oxygen species (ROS), which promote metastasis. ROS scavengers reduced metastatic potential, indicating that mtDNA mutations contribute to metastasis by increasing ROS production. These mutations also upregulate nuclear genes like MCL-1, HIF-1α, and VEGF, which are involved in angiogenesis and cell survival. The study also showed that mtDNA from high metastatic cells can induce similar effects in other cell lines, suggesting a role in metastasis. However, these mutations do not enhance tumorigenicity in non-transformed cells. The findings suggest that mtDNA mutations can influence metastasis through ROS-mediated regulation of nuclear genes, highlighting a novel function of mtDNA in cancer progression.Mitochondrial DNA (mtDNA) mutations can regulate tumor cell metastasis. This study used cybrid technology to transfer mtDNA between metastatic and non-metastatic mouse tumor cells, revealing that mtDNA mutations can enhance metastatic potential. Mutations in the ND6 gene, such as G13997A and 13885insC, cause respiratory complex I dysfunction and increased reactive oxygen species (ROS), which promote metastasis. ROS scavengers reduced metastatic potential, indicating that mtDNA mutations contribute to metastasis by increasing ROS production. These mutations also upregulate nuclear genes like MCL-1, HIF-1α, and VEGF, which are involved in angiogenesis and cell survival. The study also showed that mtDNA from high metastatic cells can induce similar effects in other cell lines, suggesting a role in metastasis. However, these mutations do not enhance tumorigenicity in non-transformed cells. The findings suggest that mtDNA mutations can influence metastasis through ROS-mediated regulation of nuclear genes, highlighting a novel function of mtDNA in cancer progression.