The cancer stem cell hypothesis may explain why conventional chemotherapies are unable to fully eradicate cancers. In this study, we examined both the prognostic and predictive significance of putative cancer stem cell markers in colorectal cancer. In this study, immunohistochemistry for three candidate cancer stem cell markers (CD133, Oct-4 and Sox-2) and for six other postulated prognostic markers (CK7, CK20, Cox-2, Ki-67, p27 and p53) were performed using tissue microarrays containing 501 primary colorectal cancer cases. Receiver-operating characteristic analysis was used to determine cut-off scores for positive protein expression. Multivariate analysis revealed that positive expression for CD133 and Oct-4 was associated with significantly worse survival in patients treated by surgery alone (P=0.023 and P<0.001, respectively) and in patients treated with 5-fluorouracil-based chemotherapy (P=0.001 and P=0.021, respectively). Stage III patients with negative CD133 expression showed an apparent survival benefit from 5-fluorouracil treatment (P=0.002), but not those with positive CD133 expression. Positive expression of CD133 was also associated with poorer clinical response to chemotherapy in stage IV patients (P=0.006). In summary, the putative cancer stem cell markers CD133 and Oct-4 showed strong prognostic significance in colorectal cancer. Our results show for the first time that CD133+ colorectal tumors are more resistant to 5-fluorouracil-based chemotherapy.