Objective To determine the killing effects of cytokine-induced killer cells bound with the bispecific antibodies for CD3 and CD133(BsAb-CIK) on CD133-high expression colorectal cancer cells. Methods The bispecific antibodies were generated by chemical coupling with anti-CD3 and anti-CD133 antibodies. CCK-8 kit was used for comparing the killing effects of CIK and BsAb-CIK on CD133-high expression colorectal cancer cell lines (SW620 and HT29) and CD133-low expression colorectal cancer cell line (LOVO). Cytokines secreted in the supernatant were detected. The xenograft for nude mice was established, and then given CIK or BsAb-CIK cells infusion via abdominal cavity. Tumor weight were compared between the groups after 1 month. Results At the different ratio of effector cells to target cells (1∶5, 1∶10, 1∶20), the cytotoxic effects of BsAb-CIK cells on CD133-high expression colorectal cancer cells (SW620 and HT29) were stronger than that of general CIK cells (P＜0.05), but there was no significant difference on CD133-low expression colorectal cancer cells (LOVO). The secretary amount of IFN-γ was higher in the supernatant of BsAb-CIK cells than the general CIK cells when killing experiments were performed. The treatment with BsAb-CIK cells resulted in a more remarkable inhibition on xenograft tumor growth than that with general CIK cells (P＜0.05). Conclusion The BsAb-CIK cells can effectively kill CD133-high expression colorectal cancer cells.