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Background: Although the exact mechanisms of nonalcoholic fatty liver disease (NAFLD) are not fully understood, numerous pieces of evidence show that the variations in mitochondrial DNA (mtDNA) level and hepatic Fibroblast growth factor 21 (FGF21) expression may be related to NAFLD susceptibility. Objectives: The main objective of this study was to determine relative levels of mtDNA copy number and hepatic FGF21 expression in a cohort of Iranian NAFLD patients and evaluate the possible relationship. Methods: This study included 27 NAFLD patients (10 with nonalcoholic fatty liver (NAFL) and 17 with non-alcoholic steatohepatitis (NASH)) and ten healthy subjects. Total RNA and genomic DNA were extracted from liver tissue samples, and then mtDNA copy number and FGF21 expression levels were assessed by quantitative real-time PCR. Results: The relative level of hepatic mtDNA copy number was 3.9-fold higher in patients than in controls ( p < 0.0001). NAFLD patients showed a 2.9-fold increase in hepatic FGF21 expression compared to controls ( p < 0.013). Results showed that hepatic FGF21 expression was positively correlated with BMI, serum ALT, and AST levels ( p < 0.05). The level of mitochondrial copy number and hepatic FGF21 expression was not significantly associated with stages of change in hepatic steatosis. Finally, there was a significant correlation between FGF21 expression and mitochondrial copy number in NAFLD patients ( p = 0.027). Conclusion: Our findings suggest a considerable rise of hepatic FGF21 mRNA levels and mtDNA-CN and show a positive correlation between them in the liver tissue of NAFLD patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Houshmand, Zeinali, Hosseini, Seifi, Danaei and Kamfar.)