Adult-onset calorie restriction (A-CR) is an experimental model of life-extension and healthy aging less explored, compared to calorie restriction begun at early ages, but one more realistic for human application. We examined the effect of A-CR on the aging rat kidney with respect to common structural age-dependent changes and the accumulation of mitochondrial enzyme abnormalities in tubular epithelial cells. A 40% calorie restriction was initiated in middle-aged rats, prior to the onset of significant age-related changes in the Fischer X Brown Norway rat kidney. This dietary intervention effectively reduced glomerulosclerosis and tubular atrophy within six months, changed the rate of interstitial fibrosis formation within one year and vascular wall thickening and the expression cytochrome c oxidase (COX)-deficient tubular epithelial cells in 18 months compared to age-matched ad libitum fed rats. Our histological approach (histochemical staining for mitochondrial enzyme activity and laser capture microdissection) coupled with mitochondrial DNA (mtDNA) PCR analyses, demonstrated that COX-deficient renal tubular epithelial cells accumulated mtDNA deletion mutations and that these cells contained unique, clonally-expanded mtDNA deletion mutations. Renal tubular epithelial cells with mitochondrial abnormalities presented cellular characteristics indicative of physiological dysfunction.