It has been proposed that the selective elimination of cancer stem cells (CSCs) using targeted therapy could greatly reduce tumor growth, recurrence, and metastasis. To develop effective therapeutic targets for CSC elimination, we aimed to define the properties of CSC mitochondria, and identify CSC-mitochondria- specific targets in colon cancer. We found that colon CSCs utilize mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP. We also found that forkhead box protein 1 (FOXM1)-induced peroxiredoxin 3 (PRDX3) maintains the mitochondrial function, and the FOXM1/PRDX3 mitochondrial pathway maintains survival of colon CSCs. Furthermore, FOXM1 induces CD133 (PROM1/prominin 1) expression, which maintains the stemness of colon CSCs. Together, our findings indicate that FOXM1, PRDX3, and CD133 are potential therapeutic targets for the elimination of CSCs in colon cancer.