Human polo-like kinase 1 (Plk1) is a key player in different stages of mitosis and modulates the spindle checkpoint at the metaphase-anaphase transition. Overexpression of Plk1 is observed in various human tumors and it is a negative prognostic factor in patients suffering from diverse cancers. We used phosphorothioate antisense oligonucleotides (ASO) targeted against Plk1, together with paclitaxel, carboplatin, and Herceptin, for the treatment of breast cancer cells to identify conditions for enhanced drug sensitivity. After transfection of the breast cancer cell lines BT-474, MCF-7, and MDA-MB-435 with Plk1-specific ASOs, paclitaxel, carboplatin, or Herceptin was added and cell proliferation, cell cycle distribution, and apoptosis were measured. Whereas the dual treatment of breast cancer cells with Plk1-specific ASOs with carboplatin or Herceptin caused only a limited antiproliferative effect in breast cancer cells, we observed synergistic effects after combination of low doses of Plk1-specific ASOs with paclitaxel, which is used in a variety of clinical anticancer regimens. Plk1-specific ASOs also acted synergistically with paclitaxel in the arrest of the cell cycle at the G(2)-M phase and in the induction of apoptosis. Interestingly, in a human xenograft experiment using MDA-MB-435 cells, the combination of Plk1 ASOs with paclitaxel led to synergistic reduction of tumor growth after 3 weeks of treatment compared with either agent alone. This study suggests that antisense inhibitors against Plk1 at well-tolerated doses may be considered as highly efficient promoters for the antineoplastic potential of taxanes, such as paclitaxel, causing synergistic effects in breast cancer cells.