We addressed the issue of whether enhanced glycolysis caused by mtDNA mutations independently induces metastasis in tumor cells using mtDNA transfer technology. The resultant trans-mitochondrial cybrids sharing the same nuclear background of poorly metastatic carcinoma P29 cells, P29mtA11 and P29mtDelta cybrids, possessed mtDNA with a G13997A mutation from highly metastatic carcinoma A11 cells and mtDNA with a 4696bp deletion mutation, respectively. The P29mtDelta cybrids expressed enhanced glycolysis, but did not express ROS overproduction and high metastatic potential, whereas P29mtA11 cybrids showed enhanced glycolysis, ROS overproduction, and high metastatic potential. Thus, enhanced glycolysis alone does not induce metastasis in the cybrids.