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Mitochondrial dysfunction with increased production of reactive oxygen species (ROS) is a characteristic feature of diabetes which is associated with damage of mitochondrial DNA (mtDNA). Alcohol metabolism generates ROS with enhanced oxidative stress leading to damage of cellular constituents including mtDNA. The aim of the present study is to investigate the impact of alcohol consumption on hepatic mtDNA damage in diabetic rats. MtDNA was isolated from hepatic tissues of non-diabetic and streptozotocin induced diabetic rats after alcohol treatment. Comprehensive screening of mtDNA displacement loop (D-loop) was carried out by PCR-Sanger’s sequencing analysis. MtDNA deletions were analyzed by long-extension PCR. Furthermore, activities of enzymatic and non-enzymatic antioxidants were measured in hepatic tissue of all rats. Our results showed increased frequency of D-loop mutations in alcoholic-diabetic rats when compared to diabetic or alcoholic non-diabetic rats. DNA mfold analysis predicted higher free energy for 15507C, 15560T and 16116C alleles compared to their corresponding wild alleles which represents less stable secondary structures with negative impact on overall mtDNA function. MtDNA deletions were observed in all experimental groups except controls. Markedly decreased activities of antioxidant enzymes viz., GPx, SOD, catalase and GSH content was identified in alcoholic-diabetic rats when compared to remaining groups. In conclusion, decreased GSH content and lowered activity of catalase, SOD and GPx favor the environment for oxidative stress, which might lead to exacerbation of hepatic mitochondrial DNA damage in diabetic rats receiving alcohol.