Abstract： Objective To explore whether or not multi-drug resistance could be reversed by RNA interference the expression of Topo Ⅱα gene in epithelial ovarian cancer cell lines in vitro. Methods (1) The best silent small interference RNA (siRNA) of Topo Ⅱα gene was designed and chose and cloned into psilencer4, 1-CMV-neo vector. The psilencer4. 1-CMV-neo-Topo Ⅱα was transfected into SKOV3/DDP cell, then Topo Ⅱα siRNA (+) SKOV3/DDP cells was incubated. (2) The Tope Ⅱα mRNA and protein expression of the stability-transfecting cell lines were detected by RT-PCR and western blot method, respectively. The resistance index, the cell cycle and the cellular content of cisplatin were detected by methyl thiazolyl tetrazolium assay, the flow cytometry and high performance liquid chromatography method before and after Topo Ⅱα RNA interference in cells. Results (1) The Topo Ⅱα gene expression level in SKOV3/DDP cells could be inhibited after the plasmid DNA psilencer4, 1-CMV-neo-Topo Ⅱα transfeced. The expression level of Tope Ⅱα mRNA in Topo Ⅱα siRNA(+)SKOV3/DDP and SKOV3/DDP cells were 0 and 0.92±0.08; the expression level of Topo Ⅱα protein in Topo Ⅱα siRNA (+) SKOV3/DDP and SKOV3/DDP cells were 0.51±0. 04 and 1.95±0.09 (P<0.01). (2) The multi-drug resistance index of Topo Ⅱα siRNA (+) SKOV3/DDP cell was significantly lower compared with that in SKOV3/DDP cell (3.46 vs 5.05, P<0.05). (3) The percentage of G_0/G_1 and G_2/M phase cell in Topo Ⅱα siRNA(+) SKOV3/DDP cells were higher than that in SKOV3/DDP cells (P<0.05). (4) The content of cisplatin in Topo Ⅱα siRNA(+)SKOV3/DDP cells treated with cisplatin for 24 hours was significantly higher than that in SKOV3/DDP cell (157.20 vs 63.99 ng, P<0.05). Conclusion The results showed that the tolerance of cisplatin would be reversed by blocking the Topo Ⅱα gene expression in cisplatin-resistant epithelial ovarian cancer cells.