Caspases are suggested to play essential roles not only in apoptotic but also in non-apoptotic functions. However, the contribution of caspases to the cell cycle regulation is unclear. Here we found that caspases including caspase-3, caspase-7, caspase-8 and caspase-9 were activated during mitosis. Chemically synthesized caspase inhibitors delayed mitotic progression and induced accumulation of mitotic cells, which exhibited abnormal chromatin condensation and incomplete chromosome segregation. Furthermore, knockdown of caspase-7 by using small interfering RNAs resulted in the inhibition of cell proliferation, but knockdown of other caspases did not show a significant effect on cell growth. The expression of short hairpin RNA directed against caspase-7 induced the cell cycle arrest at mitosis, which was rescued by the re-expression of caspase-7 containing silent mutations at the target site for the short hairpin RNA. These results revealed that caspase-7 has a novel role during cell cycle progression at mitosis.