Genistein, an isoflavone abundantly present in soybeans, possesses anticancer properties and induces growth inhibition including cell cycle arrest and apoptosis. Although abnormal cell division, such as defects in chromosome segregation and spindle formation, and polyploidization have been described, the mechanisms underlying the induction of abnormal cell division are unknown. In this study, we examined the effect of genistein on cell division in cells that are synchronized in M phase, since genistein treatment delays mitotic entry in asynchronous cells. HeLa S3 cells were arrested at the G2 phase and subsequently released into the M phase in presence of genistein. Immunofluorescence staining showed that genistein treatment delays M phase progression. Time-lapse analysis revealed that the delay occurs until anaphase onset. In addition, genistein treatment induces cleavage furrow regression, resulting in the generation of binucleated cells. Central spindle formation, which is essential for cytokinesis, is partially disrupted in genistein-treated cells. Moreover, aberrant chromosome segregation, such as a chromosome bridge and lagging chromosome, occurs through progression of cytokinesis. RhoA, which plays a role in the assembly and constriction of an actomyosin contractile ring, is delocalized from the cortex of the ingressing cleavage furrow. These results suggest that genistein treatment induces binucleated cell formation through cleavage furrow regression, which is accompanied by chromosome bridge formation and RhoA delocalization. Our results provide the mechanism that underlies genistein-induced polyploidization, which may be involved in genistein-induced growth inhibition. J. Cell. Biochem. 115: 763–771, 2014. © 2013 Wiley Periodicals, Inc.