Background: CD133 (prominin-1) is the most commonly used molecular marker of the cancer stem cells (CSCs) that maintain tumor progression and recurrence in colorectal cancer. However, the proteome of CSCs directly isolated from colorectal tumors based on CD133 expression has never been investigated. Objective: To reveal biomarkers of CD133-positive colorectal CSCs. Methods: Thirty colorectal tumor samples were collected from patients undergoing bowel resection. CD133-positive and CD133-negative cells were isolated by FACS. Comparative proteomic profiling was performed by LC-MS/MS analysis combined with label-free quantification. Verification of differentially expressed proteins was performed by flow cytometry or ELISA. CD133-knockout Caco-2 and HT-29 cell lines were generated using CRISPR-Cas9 gene editing. Results: LC-MS/MS analysis identified 29 proteins with at least 2.5-fold higher expression in CD133-positive cells versus CD133-negative cells. Flow cytometry confirmed CEACAM5 overexpression in CD133-positive cells in all clinical samples analyzed. S100A8, S100A9, and DEFA1 were differentially expressed in only a proportion of the samples. CD133 knockout in the colon cancer cell lines Caco-2 and HT-29 did not affect the median level of CEACAM5 expression, but led to higher variance of the percentage of CEACAM5-positive cells. Conclusions: High CEACAM5 expression in colorectal cancer cells is firmly associated with the CD133-positive colorectal CSC phenotype, but it is unlikely that CD133 directly regulates CEACAM5 expression.Keywords: CD133; CD66e; CEACAM5; Colorectal cancer; DEFA1; PROM1 knockout; S100A8; S100A9; cancer stem cells.