Compelling evidence has indicated a significant association between leukocyte mitochondrial DNA (mtDNA) content and incidence risks of several malignancies in a cancer-specific manner. However, to date, whether leukocyte mtDNA content can predict clinical outcome of cancer patients has never been investigated. In the present study, we measured leukocyte mtDNA content using real-time PCR-based method in a total of 598 colorectal cancer (CRC) patients and explored its prognostic values. To explore potential mechanism, we detected the immunophenotypes of peripheral blood mononuclear cells and plasma concentrations of several cytokines in CRC patients. We found that patients with high mtDNA content showed significantly worse overall survival (OS) and relapse-free survival (RFS) than those with low mtDNA content in all patient sets. Furthermore, mtDNA content and tumor node metastasis (TNM) stage exhibited a notable joint effect in prognosis prediction. Integration of TNM stage and leukocyte mtDNA content significantly improved the prognosis prediction efficacy for CRC. Importantly, patients with high mtDNA content showed OS and RFS benefits from adjuvant chemotherapy. In addition, we found that patients with high mtDNA content had a higher frequency of CD4+CD25+FOXP3+ regulatory T cells, higher plasma interleukin-2 and transforming growth factor-β1 and lower tumor necrosis factor-α concentration than those with low mtDNA content, suggesting a stronger immunosuppressive phenotype. In conclusion, our study for the first time demonstrates that leukocyte mtDNA content is an independent prognostic marker complementing TNM stage and associated with immunosuppression in CRC patients. Additionally, leukocyte mtDNA content might serve as a potential biomarker to select CRC patients who will benefit from adjuvant chemotherapy.