p 27^Kip1 (p27) acts as a tumor suppressor by inhibiting cyclin–cyclin-dependent kinase (cyclin-CDK) activity. However, mice expressing a form of p27 that is unable to bind or inhibit cyclin-CDK complexes (p27^CK–) have increased incidence of tumor development as compared with wild-type and p27^–/– mice, revealing an oncogenic role for p27. Here, we identified a phenotype of multinucleation and polyploidy in p27^CK– mice not present in p27^–/– animals, suggesting a role for p27 in G₂/M that is independent of cyclin-CDK regulation. Further analysis revealed that p27^CK– expression caused a cytokinesis and abscission defect in mouse embryonic fibroblasts. We identified the Rho effector citron kinase (citron-K) as a p27-interacting protein in vitro and in vivo and found that p27 and citron-K colocalized at the contractile ring and mid-body during telophase and cytokinesis. Moreover, overexpression of the minimal p27-binding domain of citron-K was sufficient to rescue the phenotype caused by p27^CK–. Conversely, expression of a mutant p27^CK– unable to bind citron-K did not induce multinucleation. Finally, by binding to citron-K, p27 prevented the interaction of citron-K with its activator RhoA. Taken together, these data suggest a role for p27 during cytokinesis via the regulation of citron-K activity.