Successful cytokinesis is critical for maintaining genome stability and requires the assembly of a robust central spindle to specify the cleavage furrow position, to prevent separated chromosomes from coming back together, and to contribute to midbody abscission. 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. 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 gamma-tubulin ring complexes on sucrose gradients and that XCTK2 associated with gamma-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.