Abstract Background: Mitochondria are eukaryotic organelles responsible for energy production. Quantitative changes in human mitochondrial DNA (mtDNA) copy number have been implicated in various cancer types. Data from prospective cohort studies on mtDNA copy number and colorectal cancer risk have been lacking. Methods: We evaluated the association between mtDNA copy number in peripheral blood and colorectal cancer risk in a nested case–control study of 422 colorectal cancer cases (168 cases with pre-diagnostic blood and 254 cases with post-diagnostic blood) and 874 controls who were free of colorectal cancer among participants of the Singapore Chinese Health Study. The relative mtDNA copy number was measured using real-time PCR. Unconditional logistic regression methods were employed to examine the association between mtDNA copy number and colorectal cancer risk. Results: There was a U-shaped relationship between the relative mtDNA copy number and colorectal cancer risk. Compared with the 2nd quartile, the OR (95% confidence intervals) for subjects in the lowest and highest quartiles of relative mtDNA copy numbers were 1.81 (1.13–2.89) and 3.40 (2.15–5.36), respectively (Pcurvilinearity <0.0001). This U-shaped relationship was present in both men and women, similar for colon cancer and rectal cancer, and independent of the timing of blood draw with regard to cancer diagnosis. Conclusions: This is the first prospectively designed study to show a U-shaped association between the relative mtDNA copy number and risk of colorectal cancer. Impact: The findings of the present study support that mtDNA may play a critical role in the colorectal carcinogenesis in humans. Cancer Epidemiol Biomarkers Prev; 21(9); 1574–81. ©2012 AACR.