Objectives: To investigate the role of polo-like kinase -1 (PLK1) in the first cleavage of a zygote in culture. Methods: This experiment took place in the Reproductive Medicine Center of Tongji Hospital, Wuhan, China, from 1st June 2009 to 20th November 2009. First, we detected the expression of PLK1 during the first zygotic division by using Western blotting, and then we reduced the expression of PLK1 during the first zygotic division by ribonucleic acid (RNA) interference (including 4 groups: PLK1 small interfering RNA (siRNA), siRNA control, mock transfection, and only zona pellucida (ZP) removal), finally we evaluated and compared the first cleavage rates of the 4 groups. Results: The expression of PLK1 peaked in the first M phase of zygotic cleavage (3 samples/group, 100 zygotes/sample). The relative amount of PLK1 of the mouse zygotes was reduced significantly after siRNA transfection. The first cleavage rate of the PLK1 siRNA group was significantly less than that of other groups (siRNA control, mock transfection, and only ZP removal, p=0.000). Conclusion: The PLK1 plays a crucial role during the first cleavage of one-cell embryos, and the zygotes are unable to divide successfully without functional PLK1.