Protracted mitotic arrest leads to cell death; however, the molecular signals that link these distinct processes remain poorly understood. Here we report that the pro-apoptotic BH3-only family member Bim undergoes phosphorylation in K562 cells following treatment with the microtubule targeting agents Taxol and Nocodazole. The phosphorylation of two Bim isoforms, BimEL and BimL, at the mitochondria correlates with mitotic arrest and precedes cell death induced by Taxol. It was also found that Bim undergoes transient phosphorylation during normal mitosis in K562 cells. In addition, siRNA silencing of Bim reduces sensitivity to Taxol-induced cell death. The transition of K562 cells from mitosis to G1 results in the loss of BimEL and BimL phosphorylation and correlates with the degradation of cyclin B1. The Cdk1 inhibitors, RO-3306 and Purvalanol A, block Bim phosphorylation in mitotically arrested cells. Importantly, it was found that cyclin B1 co-immunoprecipitates with endogenous Bim in mitotic extracts. Furthermore, active recombinant Cdk1/cyclin B1 phosphorylates BimEL and BimL in vitro and Serine 44 on BimL has been identified as a Cdk1 phosphorylation site. Collectively, these results suggest that Cdk1/cyclin B1-dependent hyper-phosphorylation of Bim during prolonged mitotic arrest is an important cell death signal.