BACKGROUND: Mitochondrial DNA (mtDNA) copy number in peripheral blood has been found to be associated with risk of developing several cancers. However, data on pancreatic ductal adenocarcinoma (PDAC) are very limited. METHODS: To further our knowledge on this topic, we measured relative mtDNA copy number by a quantitative real-time PCR assay in peripheral leukocyte samples of 476 PDAC cases and 357 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. RESULTS: We observed lower mtDNA copy number with advancing age (P = 6.54 × 10-5) and with a high body mass index (BMI) level (P = 0.004) and no association with sex, smoking behavior, and alcohol consumption. We found an association between increased mtDNA copy number and decreased risk of developing PDAC with an odds ratios (OR) of 0.35 [95% confidence interval (CI), 0.16-0.79; P = 0.01] when comparing the fifth quintile with the first using an unconditional logistic regression and an OR of 0.19 (95% CI, 0.07-0.52; P = 0.001) with a conditional analysis. Analyses stratified by BMI showed an association between high mtDNA copy number and decreased risk in the stratum of normal weight, consistent with the main analyses. CONCLUSIONS: Our results suggest a protective effect of a higher number of mitochondria, measured in peripheral blood leukocytes, on PDAC risk. IMPACT: Our findings highlight the importance of understanding the mitochondrial biology in pancreatic cancer.