Arsenite-induced spindle abnormalities result in mitotic cell apoptosis in several cancer cell lines, but how arsenite induces these effects is not known. Evidence to date has revealed that arsenite activates Rho guanosine triphosphatases (GTPases). Because Rho GTPases regulate spindle orientation, chromosome congression, and cytokinesis, we therefore examined the involvement of Rho GTPases and their modulators in arsenite-induced mitotic abnormalities. We demonstrated that arsenic trioxide (ATO) disrupted the positioning of bipolar mitotic spindles and induced centrosome and spindle abnormalities. ATO increased the level of the active guanosine triphosphate-bound form of Rho. Inhibition of Rho-associated protein kinases (ROCKs) by Y-27632 ameliorated ATO-induced spindle defects, mitotic arrest, and cell death. These results indicate that ATO may induce spindle abnormalities and mitotic cell death through a Rho/ROCK pathway. In addition, screening of a human kinase and phosphatase shRNA library to select genes that mediate ATO induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-biphosphate (PIP2) synthesis enzyme that belongs to the phosphatidylinositol phosphate kinase (PIPK) family. Sequestration of PIP2 by ectopic overexpression of the pleckstrin homology domain of phospholipase C-δ1 protected cells from ATO-induced cell death. Furthermore, depletion of PIP4KIIγ, but not other isoforms of the PIPK family, not only reduced Rho GTPase activation in ATO-treated cells but also alleviated ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. Thus, our results imply that ATO induces abnormalities in mitotic spindles through a PIP4KIIγ/Rho pathway, leading to apoptosis of mitotic cells.