Following fertilization the transition from a highly differentiated oocyte to a totipotent 2-cell embryo requires two unique mitotic cell cycles. The first cell cycle is characterized by a prolonged G(1) phase, DNA replication (S phase) that occurs separately in the female and male pronuclei, and a short G(2) phase that occur in the absence of cell growth. During the second cell cycle, G(1) is short whereas G(2) is prolonged and occurs concurrently with zygotic genome activation, which is essential for progression past the 2-cell stage. CDC14B, a dual specificity phosphatase that counteracts cyclin dependent kinase 1 (CDK1/CDC2A) action, regulates mitosis in somatic cells and prevents premature meiotic resumption in mouse oocytes. It is not known if CDC14B plays a role during the unique mitotic cell cycles of preimplantation development. We report that CDC14B is present in mouse embryos and localizes to mitotic centrosomes and spindles. Overexpressing CDC14B in 1-cell embryos results in 40% and 60% of the embryos arresting at the 1- and 2-cell stages, respectively. Embryos arrested at the 1-cell stage contained reduced CDC2A activity, whereas embryos arrested at the 2-cell stage were in G(2) and failed to activate the zygotic genome. In contrast, overexpressing CDC14B in meiotically-incompetent oocytes, which are arrested in a G(2)-like state and are transcriptionally active, does not repress global transcription. These data suggest that CDC14B is a negative regulator of the 1-to-2-cell transition and of zygotic genome activation in mouse embryogenesis.