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 was aimed to examine the expression of CXCR4 and CD133 protein in stage Ⅱ or Ⅲ colon cancer and related lymph nodes, and to investigate the clinical and prognostic significance in colon cancer.Immunohistochemistry 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 two proteins expression and clinicopathological parameters and the patients 5-year survival was analyzed.We have found that CXCR4 expression was detected in 74 of the 125 tumors (59.2%), and CD133 expression was detected in 45 of the 125 tumors (36.0%).Upon combining the expression of CXCR4 and CD133, co-expression (both CXCR4 and CD133 were positve) was detected in 29 of the 125 tumors (23.2%).When compared with the other combinations, co-expression of CXCR4 and CD133 proteins was significantly associated with the AJCC stage (P =0.029) and the lymph node status (P =0.020).Log-rank statistics showed that the AJCC stage (P =0.014), lymph node status (P =0.011), CXCR4 expression (P =0.023), CD133 expression (P =0.034), and co-expression of CXCR4 and CD133 proteins (P =0.003) were significant prognostic indicators for overall patients survival.We have shown that co-expression of CXCR4 and CD133 proteins is a risk factor for worse overall survival in stage Ⅱ or Ⅲ colon cancer patients,indicating that co-expression of CXCR4 and CD133 proteins might contribute to the progression of colon cancer.