Rho GDP-dissociation inhibitors (RhoGDIs) are regulators of Rho family GTPases. RhoGDIβ has been implicated in cancer progression, but its precise role remains unclear. We determined the subcellular localization of RhoGDIβ and examined the effects of its overexpression and RNAi knockdown in cancer cells. Immunofluorescence staining showed that RhoGDIβ localized to centrosomes in human cancer cells. In HeLa cells, exogenous GFP-tagged RhoGDIβ localized to centrosomes and its overexpression caused prolonged mitosis and aberrant cytokinesis in which the cell shape was distorted. RNAi knockdown of RhoGDIβ led to increased incidence of monopolar spindle mitosis resulting in polyploid cells. These results suggest that RhoGDIβ has mitotic functions, including regulation of cytokinesis and bipolar spindle formation. The dysregulated expression of RhoGDIβ may contribute to cancer progression by disrupting these processes.