Abstract Objective. Chemotherapy is highly effective in treating a number of gynecologic malignancies; however, its effectiveness diminishes with repeated exposures due to the emergence of multi-drug resistance (MDR). The aim of this study was to establish a permanent MDR gene knockdown model via infection with the siRNA-hairpin expression vector. The impact of transfecting the RNAi upon MDR-1 mRNA and P-glycoprotein expression as well as resultant chemotherapy resistance was assessed. Methods. Multi-drug resistant cell line MES-SA/DX5 was transfected with the siRNA-hairpin expression vector (pSMDR-HYG) designed to target MDR-1 mRNA. A negative control was established utilizing a vector lacking the anti-sense component (pSCON-HYG). The LD 50 of doxorubicin for the stable transfectants was determined utilizing a cytotoxic MTT assay. The mRNA expression of MDR-1 gene among those cell lines was evaluated by semi-quantitative RT-PCR. The product of P-glycoprotein (P-gp) was examined by Western blotting hybridization and immunostaining. Results. Two stable transfected cell lines: MES-SA/DX5-M (with pSMDR-HYG) and MES-SA/DX5-C (with pSCON-HYG) were established. The cell line MES-SA/DX5-M was nearly 7 times more sensitive to doxorubicin than MES-SA/DX5-C and its parent cell line MES-SA/DX5 ( P < 0.01). The mRNA expression of the MDR-1 gene in MES-SA/DX5-M was also statistically significantly lower than in the other 2 cell lines ( P < 0.01) as assessed by semi-quantitative RT-PCR. A barely detectable signal for P-gp (170 kDa) was observed in MES-SA/DX5-M. The vast majority of MES-SA/DX5-M cells were immunohistochemically negative for P-gp. Conclusions. Stable, sequence-specific MDR-1 gene silencing can be demonstrated by inducing the endogenous expression of hairpin siRNA. Hairpin-siRNA-based MDR-1 gene silencing correlated with decreased levels of MDR-1 mRNA and P-gp, thereby restoring permanent native chemosensitivity. This methodologic strategy may have significant clinical impact in reversing chemo-resistance, especially the multi-drug-resistant phenotype, in the treatment of gynecologic malignancies.