Abstract: Objective To screen human breast cancer cell line of MDR1 gene silenced and compare its biological properties. Methods The lentiviral vector of expressing MDR1 gene short hairpin RNA (shRNA ) was produced by BLOCK-iT Lentiviral RNAi Expression System and transducted into human breast cancer cell line MCF-7 and corresponding doxorubicin-resistant human breast cancer cell line MCF-7/ADM, which were selected subsequently with Blasticidin and MDR1 gene silenced cells including MCF-7/RNAi and MCF-7/ADM/RNAi were obtained. MDR1 mRNA level was measured by quantitative RT-PCR. The expression and function of P-glycoprotein(P-GP) were measured by flow cytometry.The drug sensitivity was evaluated by MTT assay.Results Two stably transducted selected cell lines, MCF-7/RNAi and MCF-7/ADM/RNAi, were obtained after being selected with 10μg/ml Blasticidin for 12 days. The relative expression levels of MDR1 mRNA in four cell lines,including MCF-7、MCF-7/RNAi、MCF-7/ADM and MCF-7/ADM /RNAi were 1、0.13、17.14 and 2.01 respectively; P-GP expression levels were (1.5±0.3）％、（1.2±0.2）％、（89.4±3.6）％ and（16.3±1.9）％, respectively; the retention of cellular rhodamine 123 were （92.4±3.1）％、（90.6±4.0）％、（13.6±1.6）％、and（72.4±2.8）％,respectively.The IC50 of four cell lines for ADM were 0.90、0.92、19.61 and 4.04mg/ml,respectively.Conclusions The data indicate that MDR1 gene stably silenced by lentivirals vector can effectively reverse multidrug resistance of human breast cancer cells.