Abstract: Objective To explore the effect of shRNA targeted to multidrug resistance gene 1(MDR1) by RNA interference (RNAi) on drug resistance of doxorubicin-resistant human leukemia cell line K562/ADM. Methods The RNAi plasmid of pENTRTM/U6-MDR1,targeting to human MDR1 gene, was transfected into doxorubicin-resistant human leukemia cell line K562/ADM and parental cell line K562.After 48h transfection, MDR1mRNA level was determined by real time RT-PCR. The expression and function of P-gp protein was analyzed by flow cytometry. Drug resistance of cells was detected by MTT assay. Results Compared with non-transfected cells, MDR1mRNA and expression levels and function ofP -gp protein in cells of pENTRTM/U6-MDR1 group decreased significantly（ P <0.05）.The resistance on doxorubicin was decreased drmatically（ P <0.05）. Conclusion The resistance on doxorubicin of human doxorubicin-resistant leukemia cells was reversed by MDR1 gene down-regulating.