Colon cancer stem cells (CCSCs) play an important role in facilitating colon cancer occurrence, metastasis and drug resistance. The results of our previous studies confirmed that the well-studied antioxidant gene peroxiredoxin-2 (PRDX2) promotes colon cancer progression. However, the underlying function and mechanisms associated with PRDX2 remodeling in the context of CCSCs have remained poorly studied. In our present study, we demonstrated that PRDX2 is highly expressed in CD133/CD44-positive colon cancer tissues and spheroid CD133+CD44+ CCSCs. PRDX2 overexpression was shown to be closely correlated with CD133+CD44+ CCSCs in colon cancer. Furthermore, PRDX2 depletion markedly suppressed CD133+CD44+ CCSC stemness maintenance, tumor initiation, migration and invasion and liver metastasis. Furthermore, the expression of various EMT markers and Wnt/β-catenin signaling proteins was altered after PRDX2 inhibition. In addition, PRDX2 knockdown led to increased ROS production in CD133+CD44+ CCSCs, sensitizing CCSCs to oxidative stress and chemotherapy. These results suggest that PRDX2 could be a possible therapeutic target in CCSCs.