Vanadocene dichloride (VDC), a vanadium containing metallocene dihalide exhibits promising anticancer activity. However, its mechanism of action remains elusive as several diverse targets and pathways have been proposed for its anticancer activity. In this study, we observed that VDC inhibited the proliferation of mammalian cancer cells and induced apoptotic cell death by altering the mitochondrial membrane potential and the expression of bcl2 and bax. Probing further into its anticancer mechanism, we found that VDC caused depolymerization of interphase microtubules and blocked the cells at mitosis with considerable proportion of cells exhibiting monopolar spindles. The reassembly of cold depolymerized microtubules was strongly inhibited in the presence of 10 μM VDC. VDC perturbed the microtubule-kinetochore interactions during mitosis as indicated by the absence of cold stable spindle microtubules in the cells treated with 20 μM VDC. Using goat brain tubulin, we found that VDC inhibited the steady-state polymer mass of microtubules and bound to tubulin at a novel site with a K of 9.71 ± 0.19 μM and perturbed the secondary structure of tubulin dimer. In addition, VDC was also found to bind to the mitotic kinesin Eg5 and inhibit its basal as well as microtubule stimulated ATPase activity. The results suggest that disruption of microtubule assembly dynamics and inhibition of the ATPase activity of Eg5 could be a plausible mechanism for the antiproliferative and antimitotic activity of VDC.Graphic abstract.