AIM: To investigate the effect of 5 - fluorouracil ( 5 - FU ) on the expression of the stem cell marker CD133 on colon cancer stem cells. METHODS:CD133 expression on several colon cancer cell lines was detected by flow cytometry. The CD133 positive cells from DLD1 cells were separated by the method of magnetic activated cell separation. Colony assay was used to measure self - renew ability and MTS assay was used to detect the sensitivity to 5 - FU after separation. After 5 - FU treatment, the change of CD133 mRNA level was measured by qPCR. RESULTS: CD133 expression on the surface of colon cacner cell lines DLD1, HT29, SW480, HCT116, Lovo, RKO was 30.20% , 82.00% , 0.34% , 91.80% , 85.30% , 0.28% respectively. DLD1 cells had two obvious populations according to CD133 expression. CD133 positive cells were separated from DLD1 cells, the positive purity was 87.21% ±5.33% and the negative purity was 84.30% ±4.65%. CD133 positive cells formed more colonies with limited dilution colony assay (46.33% ±4.44% vs 31.00% ±2.00% , P <0.05). CD133 positive cells were less sensitive to 5 - FU compared to CD133 negative cells(20% less, P <0.01). 5 - FU at concentration of 1 mg/L upregulated CD133 mRNA expression in both DLD1 and HT29 cells, the relative quantity was increased from 1 to 1.684 ±0.012(P <0.01 )and 30.702 ±0.280 to 49.379 ±0.460(P <0.01) in HT29 and DLD1, respectively. CONCLUSION: Compared to CD133 negative cells, CD133 positive cells show more ability to form colonies in vitro, and are less sensitive to 5 - FU. 5 - FU upregulats the mRNA expression of CD133, resulting in the CD133 colon cancer stem cells enrichment during 5 - FU treatment.