According to the “mitochondrial theory of aging” the lifelong accumulation of various kinds of damage to mitochondrial DNA (mtDNA) has been related to the age-dependent mitochondrial bioenergetic dysfunction. Caloric restriction (CR) diet is able to prevent or delay the onset of several age-related damages to mtDNA. The effects of aging and CR on the presence of abasic sites and single-strand breaks of the sugar–phosphate backbone in mtDNA have been analyzed by applying Ligation Mediated-PCR to a H strand region of brain mtDNA from young and old ad libitum-fed and old CR-treated rats. The region, encompassing the Direct Repeat 1 of the 4,834 bp-long deletion, is highly damaged in the old ad libitum-fed animals with respect to the young ones, whereas in the CR rats it shows a much lower extent of damage. The data confirm, at single nucleotide resolution, the protective effect of CR on the age-related mtDNA damage.