We previously reported that p53 plays as a key regulator in tetraploid G1 checkpoint, which is activated by actin damage-induced cytokinesis blockade and then prevents uncoupled DNA replication and nuclear division without cytokinesis. In this study, we investigated a role of Skp2, which targets CDK2 inhibitor p27/Kip1, in actin damage-induced tetraploid G1 arrest. Expression of Skp2 was decreased, but p27/Kip1 was increased after actin damage-induced cytokinesis blockade. The roles of Skp2 repression in tetraploid G1 arrest was investigated by analyzing the effects of ectopic expression of Skp2. After actin damage, ectopic expression of Skp2 resulted in DNA synthesis and accumulation of multinucleated cells, and eventually induced apoptosis. These results suggest that Skp2 repression is important for sustaining the tetraploid G1 arrest after cytokinesis blockade and required to prevent uncoupled DNA replication and nuclear division without cytokinesis.