Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of combined antiretroviral therapy (cART) and are widely used in anti-human immunodeficiency virus (HIV) therapy. Long-term administration of NRTIs can result in mitochondrial dysfunction in certain HIV-1-infected patients. However, NRTI-associated liver mitochondrial toxicity is not well known. Herein, the liver autopsy of acquired immune deficiency syndrome (AIDS) patients and the liver tissues of mice with 12 months of NRTI exposure were used to identify NRTI-associated liver toxicity with immunofluorescence, quantitative real-time polymerase chain reaction (qPCR), Amplex red and horseradish peroxidase, and cloning and sequencing. Laser capture microdissection was used to capture hepatocytes from liver tissues. We observed DNA oxidative damage and mitochondrial DNA (mtDNA) loss in the livers of AIDS patients, and cART patients had higher DNA oxidative damage and lower DNA repair function in liver tissues than non-cART patients. We also observed liver oxidative damage, increased DNA repair and mtDNA loss in mice with exposure to four different NRTIs for 12 months, and hepatocytes had no more mtDNA loss than liver tissues. Although NRTIs could induce mitochondrial hydrogen peroxide production, increased mitochondrial oxygen consumption was found with a Clark-type electrode. The captured hepatocytes had greater diversity in their mtDNA D-loop, dehydrogenase subunit1 (ND1) and ND4 than the controls. Long-term NRTI exposure induced single nucleotide variation in hepatocellular mtDNA D-loop, ND1 and ND4. Our findings indicate that NRTIs can induce liver mtDNA lesions, but simultaneously enhance mitochondrial function and mtDNA repair.