Although CXCR4 and CD133 have been implicated in the metastatic process of malignant tumors, the clinicopathological significance of their expression in human colon cancer is not fully understood. The present study aimed to examine the expression of the CXCR4 and CD133 proteins in cases of stage II or III colon cancer and the related lymph nodes and to investigate the clinical and prognostic significance of these proteins in colon cancer. Immunohistochemical analysis was performed to examine CXCR4 and CD133 protein expression in paraffin-embedded stage II or III primary colon cancer tissues and matched lymph nodes. The correlation between the expression of the two proteins and clinicopathological parameters and the patient 5-year survival was analyzed. CXCR4 expression was detected in 74 of the 125 tumors (59.2%) and CD133 expression was detected in 45 (36.0%). The co-expression of CXCR4 and CD133 (both CXCR4 and CD133 were positive) was detected in 29 of the 125 tumors (23.2%). Compared with the other combinations, the co-expression of the CXCR4 and CD133 proteins was significantly associated with American Joint Committee on Cancer (AJCC) stage (P=0.029) and lymph node status (P=0.020). Log-rank analysis revealed that AJCC stage (P=0.014), lymph node status (P=0.011), CXCR4 expression (P=0.023), CD133 expression (P=0.034) and the co-expression of the CXCR4 and CD133 proteins (P=0.003) were significant prognostic indicators for the overall survival of patients. The results of the present study show that the co-expression of the CXCR4 and CD133 proteins is a risk factor for poor overall survival in stage II or III colon cancer patients, indicating that the co-expression of the CXCR4 and CD133 proteins contributes to the progression of colon cancer.