Traditional antimitotic drugs for cancer chemotherapy often have undesired toxicities to healthy tissues, limiting their clinical application. Developing novel agents that specifically target tumor cell mitosis is needed to minimize the toxicity and improve the efficacy of this class of anticancer drugs. We discovered that mdivi-1 (mitochondrial division inhibitor-1), which was originally reported as an inhibitor of mitochondrial fission protein Drp1, specifically disrupts M phase cell cycle progression only in human tumor cells, but not in non-transformed fibroblasts or epithelial cells. The antimitotic effect of mdivi-1 is Drp1 independent, as mdivi-1 induces M phase abnormalities in both Drp1 wild-type and Drp1 knockout SV40-immortalized/transformed MEF cells. We also identified that the tumor transformation process required for the antimitotic effect of mdivi-1 is downstream of SV40 large T and small t antigens, but not hTERT-mediated immortalization. Mdivi-1 induces multipolar mitotic spindles in tumor cells regardless of their centrosome numbers. Acentrosomal spindle poles, which do not contain the bona-fide centrosome components γ-tubulin and centrin-2, were found to contribute to the spindle multipolarity induced by mdivi-1. Gene expression profiling revealed that the genes involved in oocyte meiosis and assembly of acentrosomal microtubules are highly expressed in tumor cells. We further identified that tumor cells have enhanced activity in the nucleation and assembly of acentrosomal kinetochore-attaching microtubules. Mdivi-1 inhibited the integration of acentrosomal microtubule-organizing centers into centrosomal asters, resulting in the development of acentrosomal mitotic spindles preferentially in tumor cells. The formation of multipolar acentrosomal spindles leads to gross genome instability and Bax/Bak-dependent apoptosis. Taken together, our studies indicate that inducing multipolar spindles composing of acentrosomal poles in mitosis could achieve tumor-specific antimitotic effect, and mdivi-1 thus represents a novel class of compounds as acentrosomal spindle inducers (ASI).