- Kinesin-5 motors fulfill essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT)-plus-end directed motors. The Saccharomyces cerevisiae kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here we have examined Cin8 directionality control using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly towards the midzone, in the plus-end direction of the interpolar MTs. Occasionally, Cin8 also moved faster towards the spindle poles, in the minus-end direction of the MTs. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel microtubules, while driving steady plus-end relative sliding. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic-strength dependent directional switching of Cin8 in vitro. In vivo, the deletion mutant exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.