Abstract: Objective To observe the autophagy of CD133+ CSCs induced by 5-FU,and explore the effect of autophagy on the chemotherapy Methods The cell proliferative inhibition rates and IC50 of 5-FU on human colon cancer cell lines SW480 were measured by MTT assay. CD133+ CSCs were purified with MACS(magnetic activated cell-sorting system) CD133 cell isolation kit. Then the morphplpgical features of cells were observed using transmission election microscopy(TEM),and the autophagic vacuoles(AV) was observed using fluorescent microscope by monodansylcadaverin(MDC) staining. The effect of autophagy on the chemotherapey was evaluated by cell viability assay and colony-forming test. Results The vibility of human colon cancer cell line SW480 was obviously inhibited with different contrations of 5-FU,half of inhibition concentration(IC50) value was (1.09±0.18)μg/mL .Autophagsomes and autophagy vesica were observed in CD133+ CSCs. The combination treatment of 5-FU and 3-MA significantly decreased the viability of CD133+CSCs from (81.8±4.6)% to (56.3±5.5)%(P<0.05), and the colony-forming efficiency from (81.1±3.4)% to (64.4±4.8)% (P<0.05), compared with 5-FU alone. Conclusion 5-FU may induce autophagy in CD133+ CSCs. Inhibition of autophagy may be employed to increase the cytotoxic effect of CD133+ CSCs to 5-FU.