Successful cytokinesis is critical for maintaining genome stability [1, 2] and requires the assembly of a robust central spindle to specify the cleavage furrow position [3], to prevent separated chromosomes from coming back together [4], and to contribute to midbody abscission [5, 6]. A proper central spindle is assembled and maintained by a number of microtubule-associated and molecular motor proteins that sort microtubules into bundles with their plus ends overlapping at the center [1, 2]. The mechanisms by which different factors organize the central spindle microtubules remain unclear. We found that perturbation of the minus-end-directed Kinesin-14 HSET increased the frequency of polyploid cells, which resulted from a failure in cytokinesis. In addition, HSET knockdown resulted in severe midzone microtubule organization, most notably at microtubule minus ends, as well as mislocalization of several midbody-associated proteins. Biochemical analysis showed that both human HSET and Xenopus XCTK2 cofractionated with the γ-tubulin ring complexes on sucrose gradients and that XCTK2 associated with γ-tubulin and Xgrip109 by immunoprecipitation. Our data reveal the novel finding that a minus-end-directed motor contributes to the organization and stability of the central spindle, which is needed for proper cytokinesis.