Centralspindlin is essential for the formation of microtubule bundle structures and the equatorial recruitment of factors critical for cytokinesis. Stable accumulation of centralspindlin at the spindle midzone requires its multimerization into clusters and Aurora B kinase activity, which peaks at the central spindle during anaphase. Although Aurora B phosphorylates centralspindlin directly, how this regulates centralspindlin localization is unknown. Here we identify a novel regulatory mechanism by which Aurora B enables centralspindlin to accumulate stably at the spindle midzone. We show that 14-3-3 protein binds centralspindlin when the kinesin-6 component MKLP1 is phosphorylated at S710. 14-3-3 prevents centralspindlin from clustering in vitro, and an MKLP1 mutant that is unable to bind 14-3-3 forms aberrant clusters in vivo. Interestingly, 14-3-3 binding is inhibited by phosphorylation of S708, a known Aurora B target site that lies within the motif bound by 14-3-3. S708 phosphorylation is required for MKLP1 to stably localize to the central spindle, but it is dispensable in an MKLP1 mutant that does not bind 14-3-3. We propose that 14-3-3 serves as a global inhibitor of centralspindlin that allows Aurora B to locally activate clustering and the stable accumulation of centralspindlin between segregating chromosomes.