Commonly used drugs for the treatment of breast cancer patients like paclitaxel and Herceptin often show severe side effects or induce resistance in clinical settings. Thus, we analysed a combination of Plk1 (polo-like kinase 1)-specific small interfering RNAs (siRNAs), a powerful tool to induce ‘mitotic catastrophe’in cancer cells, together with these drugs to identify conditions for enhanced drug sensitivity. After transfection, the antineoplastic agents were added and cell proliferation, apoptosis and cell cycle distribution in breast cancer cells (MCF-7, SK-BR-3, MDA-MB-435 and BT-474) and in primary human mammary epithelial cells were determined. Downregulation of cellular Plk1 levels led to an elevated percentage of cells in G 2/M phase. The percentage of apoptotic nuclei in MCF-7, MDA-MB-435, SK-BR-3 and BT-474 cells was clearly increased after incubation with Plk1-specific siRNAs and paclitaxel. Interestingly, the caspase pathway was activated after treatment with Plk1-specific siRNAs and paclitaxel or Herceptin. Treatment of breast cancer cells with siRNAs targeting Plk1 improved the sensitivity toward paclitaxel and Herceptin in a synergistic manner. In all experiments, very low concentrations across a wide range of clinically relevant concentrations were sufficient to induce an antiproliferative effect. The combination of Plk1-specific siRNAs with modern breast cancer drugs seems to represent rational combinations to be tested in preclinical trials.