The segregation of the genetic material during mitosis is coordinated by the mitotic spindle, whose mechanism of action depends upon the polarity patterns of its constituent microtubules (MTs). Homotetrameric mitotic kinesin-5 motors are capable of crosslinking and sliding adjacent spindle MTs, but it is unknown if they, or other motors, contribute to the establishment of these MT polarity patterns. Here we explored if the Drosophila embryo kinesin-5, KLP61F, which is thought to crosslink both parallel and anti-parallel MTs, displays a preference for the parallel or anti-parallel orientation of MTs. In motility assays, KLP61F was observed to crosslink and slide adjacent MTs, as predicted. Remarkably, KLP61F displayed a three-fold higher preference for crosslinking MTs in the antiparallel, relative to the parallel orientation. This polarity preference was observed in the presence of ADP or in ATP plus AMPPNP, but not in AMPPNP alone, which induces instantaneous rigor binding. Also, a purified motorless tetramer containing the C-terminal tail domains displayed an antiparallel orientation preference, confirming that motor activity is not required. The results suggest that, during the morphogenesis of the Drosophila embryo mitotic spindle, the crosslinking and sliding activities of KLP61F could facilitate the gradual accumulation of KLP61F within antiparallel interpolar (ip) MTs at the equator, where the motor could then generate force to drive poleward flux and pole-pole separation.