Abstract: Objective To construct the eukaryotic expression vector pIRES-IL-24-TRAIL and investigate its infl uences on the migration and apoptosis of breast cancer stem cells. Methods The total mRNA isolated from human placenta tissue was used as template and reverse transcriptase-polymerase chain reaction(RTPCR) was applied. cDNA fragment encoding human TRAIL gene was amplified and cloned into pIRES vector. cDNA fragment encoding human IL-24 gene was amplifi ed and cloned into the recombinant eukaryotic expression vector pIRES-TRAIL to construct pIRES-IL-24-TRAIL plasmid after sequencing. Subsequently，plasmid DNA of pIRES-IL-24-TRAIL was transfected into breast cancer stem cells MCF-7 and MDAMB-231 mediated by Lipofectamine2000 transfection reagent. Cell apoptosis was measured with flow cytometry. Wound healing test was performed for cell motility assay. Results (1)cDNA sequence encoding human TRAIL and IL-24 gene was successfully cloned，and recombinant eukaryotic expression vector pIRES-IL-24-TRAIL was constructed.(2)The apoptosis rate in the transfected group tested by fl ow cytomtry was higher than that in the control group(P<0.05).(3)Wound-healing assay showed that cell migration in the transfected group could be more effectively suppressed than that in the control group(P<0.05). Conclusion pIRES-IL-24-TRAIL is constructed successfully and could promote the apoptosis of breast cancer stem cells and impede tumor cell migration.