High-fructose corn syrup (HFCS) is consumed worldwide. However, it has been demonstrated that an increased intake of sweetened beverages, including those sweetened using fructose, is associated with the development of childhood obesity. It is unknown why the negative effects of fructose are stronger in young persons than in elderly individuals. In recent years, mitochondria have been identified as 1 of the targets of the negative effects of fructose; they possess their own genome called mitochondrial DNA (mtDNA), which encodes genes involved in metabolic functions. We hypothesized that HFCS intake affects mtDNA in the livers of rats, and that the intensity of these effects is age-dependent. The experimental period was divided into 3 parts: childhood and adolescence (postnatal day [PD] 21-60), young adulthood (PD61-100), and adulthood (PD101-140). Rats in the different age groups were assigned to receive either water (control group [CONT]) or a 20% HFCS solution (HFCS). The hepatic mtDNA copy number of the HFCS group was higher than that of the CONT group in childhood and adolescence. In addition, the mtDNA methylation level was increased in the HFCS group in the same experimental period. No significant differences were observed between the CONT and HFCS groups during the other experimental periods. We demonstrated that HFCS has the strongest effect on mtDNA during childhood and adolescence, suggesting a need to analyze the HFCS intake of young people.