Abstract: Objective To obtain cancer stem cells (CSCs) from surgically resected colorectal cancer specimens and identify their stem cell characteristics. Methods Colorectal cancer tissue specimen obtained from a patient undergoing radical resection of colorectal cancer were implanted in nude mice, and the xenograft was harvested 1 month later to obtain purified tumor cells by enzyme digestion and adherent culture. The CSCs were screened by limiting dilution method and serum-free culture to identify their phenotypes. Soft agar colony assay was used to assess the proliferative ability of the CSCs and human colorectal cancer cell line SW480. The tumorigenic ability of the isolated CSCs and SW480 cells was evaluated by observing their subcutaneous tumor formation in nude mice. Western blotting and immunofluorescence assay were used to detect the immunophenotype of the CSCs and SW480 cells. Results The primary cultured CSCs from clinical specimens of colorectal cancer underwent differentiation in the presence of serum in the culture. Soft agar colony formation assay showed that the CSCs had a colony formation rate above 50%, significantly higher than the rate of colorectal cancer SW480 cells (4.41%; P<0.01). In nude mice, subcutaneous injection of 500 CSCs was sufficient to result in subcutaneous tumor formation, while the injection of 500 SW480 cells failed to form any subcutaneous tumors. The CSCs expressed CD133 and CD44 but not CK7, while SW480 cells expressed CK7 but not CD133 or CD44. Conclusion CSCs can be derived by primary culture of cancer cells obtained from surgically resected colorectal cancer tissue followed by serum-free culture, and the CSCs obtained have self-renewal and differentiation abilities.