Antimitotics such as taxanes are being considered as alternatives to conventional cisplatin-based chemotherapy in patients with bladder cancer, but the molecular determinants of sensitivity or resistance to these agents in bladder cancer cells have not been defined. Here we examined the cytotoxic effects of a novel antimitotic, the Eg5 inhibitor AZD4877, in a molecularly diverse panel of human bladder cancer cell lines. The cells displayed heterogeneous responses to the drug that correlated closely with sensitivity to docetaxel but not with sensitivity to cisplatin. Global gene expression profiling identified p63 as the top gene that was differentially expressed between sensitive and resistant cell lines. Stable knockdown of p63 inhibited cell death induced by either AZD4877 or docetaxel and was associated with decreased proliferation and decreased expression of c-myc. Furthermore, c-myc knockdown also rendered cells resistant to AZD4877 or docetaxel. Together, our results implicate p63 and its downstream target c-myc as determinants of sensitivity to anti-mitotics in bladder cancer cells. Our data also suggest that anti-mitotics and cisplatin target different subsets of bladder cancer cells, a conclusion that may have important implications for the therapy of muscle-invasive bladder cancers.