AbstractMitochondrial DNA (mtDNA) mutations are found in many types of cancers and suspected to be involved in carcinogenesis, although the mechanism has not been elucidated. In this study, we report that consecutive C-to-T mutations (hypermutations), a unique feature of mutations induced by APOBECs, are found in mtDNA from cervical dysplasia and oropharyngeal cancers. In vitro, we found that APOBEC3A (A3A) and 3B (A3B) expression, as well as mtDNA hypermutation, were induced in a cervical dysplastic cell line W12 when cultured in a differentiating condition. The ectopic expression of A3A or A3B was sufficient to hypermutate mtDNA. Fractionation of W12 cell lysates and immunocytochemical analysis revealed that A3A and A3B could be contained in mitochondrion. These results suggest that mtDNA hypermutation is induced upon keratinocyte differentiation, and shed light on its molecular mechanism, which involves A3s. The possible involvement of mtDNA hypermutations in carcinogenesis is also discussed.