Cytokinesis mediates separation of daughter cells at the end of cell division. We have developed a high-throughput approach for monitoring cell-autonomous cytokinesis in non-adherent cells. Focusing on cytokinesis termination, we show that chemical inhibition of protein phosphatase 1 (PP1) and PP2A specifically in late cytokinesis activates cytokinesis regression, which is distinct from any known cytokinesis failure, and is not a by-product of abnormal furrow ingression or chromatin bridges. This process is characterized by the formation of cortical blebs primarily at the intercellular bridge, reopening of the cleavage furrow and reassembly of an interphase-like microtubule network, but not by chromatin recondensation and mitotic spindle formation. Finally, cytokinesis regression is suppressed by chemical inhibition of aurora kinases but not Cdk1 or PLK1. Altogether, our results highlight a hitherto uncharacterized facet of the counter-activity of PP1/PP2A and aurora kinases in the final step of cell division, which ultimately secure the conclusion of cytokinesis, thereby preventing polyploidy and genomic instability.