In accordance with the hypothesis that cancer formation is a process of somatic evolution driven by natural selection, signature of positive selection has been detected on a number of cancer-related nuclear genes. It remains, however, controversial whether a similar selective pressure has also acted on mitochondrial DNA (mtDNA), a small molecule in mitochondrion that may play an important role in tumorigenesis by altering oxidative phosphorylation. To better understand the mutational pattern on cancerous mtDNA and decipher the genetic signature left by natural selection, a total of 186 entire mitochondrial genomes of cancerous and adjacent normal tissues from 93 esophageal cancer patients were obtained and extensively studied. Our results revealed that the observed mutational pattern on the cancerous mtDNAs might be best explained as relaxation of negative selection. Taking into account an additional 1,235 cancerous (nearly) complete mtDNA sequences retrieved from the literature, our results suggested that the relaxed selective pressure was the most likely explanation for the accumulation of mtDNA variation in different types of cancer. This notion is in good agreement with the observation that aerobic glycolysis, instead of mitochondrial respiration, plays the key role in generating energy in cancer cells. Furthermore, our study provided solid evidence demonstrating that problems in some of the published cancerous mtDNA data adequately explained the previously contradictory conclusions about the selective pressure on cancer mtDNA, thus serving as a paradigm emphasizing the importance of data quality in affecting our understanding on the role of mtDNA in tumorigenesis.