Overexpression of the oncogenic serine/threonine kinase Pim-1 has been shown to induce chromosomal missegregation and polyploidy in prostate epithelial cell lines (1). Here we demonstrated that Pim-1-induced polyploidy develops in a passage-dependent manner in culture consistent with a stochastic mode of progression. Induction of chromosomal instability by Pim-1 was not restricted to prostate cells as it was also observed in telomerase-immortalized normal human mammary epithelial cells. Elevated levels of cyclin B1 protein, but not its messenger RNA, were evident in early passage Pim-1 overexpressing cells, suggesting that increased cyclin B1 levels contribute to the development of polyploidy. Furthermore, regulation of cyclin B1 protein and cyclin B1/CDK1 activity after treatment with anti-microtubule agents was impaired. Small interfering RNA targeting cyclin B1 reversed the cytokinesis delay but not the mitotic checkpoint defect in Pim-1 overexpressing cells. These results indicated that chronic Pim-1 overexpression dysregulates cyclin B1 protein expression, which contributes to the development of polyploidy by delaying cytokinesis.