An increase in the rate of glycolysis is one of the metabolic alterations in most cancer cells. However, the role of alterations in mitochondrial function and mitochondrial DNA (mtDNA) in carcinogenesis still remains unclear. In this study, we analyzed the nucleotide sequence of the D-loop and the copy number of mtDNA in 54 hepatocellular carcinomas (HCCs), 31 gastric, 31 lung, and 25 colorectal cancers as well as their corresponding non-tumorous tissues. The results revealed that 42.6% (23/54) of the HCCs, 51.6% (16/31) of the gastric cancers, 22.6% (7/31) of the lung cancers, and 40.0% (10/25) of the colorectal cancers harbored mutation(s) in the D-loop of mtDNA. The mtDNA mutations in 43.5% (10/23) of the HCCs, 62.5% (10/16) of the gastric cancers, 57.1% (4/7) of the lung cancers, and 90.0% (9/10) of the colorectal cancers were changes in the mononucleotide or dinucleotide repeats, deletions, or multiple insertions. Moreover, we found that there is a significant decrease in mtDNA copy number in 57.4% (31/54) of the HCCs, 54.8% (17/31) of the gastric cancers, 22.6% (7/31) of the lung cancers, and 28.0% (7/25) of the colorectal cancers compared with the corresponding non-tumorous tissues. It is noteworthy that the incidence of somatic mutations in the D-loop of mtDNA in the cancers of later stages was higher than that of the early-stage cancers. Taken together, our findings suggest that instability in the D-loop region of mtDNA, together with the decrease in mtDNA copy number, is involved in the carcinogenesis of human cancers.