Mitochondrial alteration has been long proposed to play a major role in tumorigenesis. Recently, mitochondrial DNA (mtDNA) mutations have been found in a variety of cancer cells. In this study, we examined the contribution of mtDNA mutation and mitochondrial dysfunction in tumorigenesis first using human cell lines carrying a frame-shift at NADH dehydrogenase (respiratory complex I) subunit 5 gene (ND5); the same homoplasmic mutation was also identified in a human colorectal cancer cell line earlier. With increasing mutant ND5 mtDNA content, respiratory function including oxygen consumption and ATP generation through oxidative phosphorylation declined progressively, while lactate production and dependence on glucose increased. Interestingly, the reactive oxygen species (ROS) levels and apoptosis exhibited antagonistic pleiotropy associated with mitochondrial defects. Furthermore, the anchorage-dependence phenotype and tumor-forming capacity of cells carrying wild-type and mutant mtDNA were tested by growth assay in soft agar and subcutaneous implantation of the cells in nude mice. Surprisingly, the cell line carrying the heteroplasmic ND5 mtDNA mutation showed significantly enhanced tumor growth, while cells with homoplasmic form of the same mutation inhibited tumor formation. Similar results were obtained from the analysis of a series of mouse cell lines carrying a nonsense mutation at ND5 gene. Our results indicate that the mtDNA mutations might play an important role in the early stage of cancer development, possibly through alteration of ROS generation and apoptosis.