Neurons polarize to form elaborate multiple dendrites and one long axon. The establishment and maintenance of axon/dendrite polarity are fundamentally important for neurons. Recent studies have demonstrated that the polarity complex PAR-3-PAR-6-atypical protein kinase C (aPKC) is involved in polarity determination in many tissues and cells. The function of the PAR-3-PAR-6-aPKC protein complex depends on its subcellular localization in polarized cells. PAR-3 accumulates at the tip of growing axons in cultured rat hippocampal neurons, but the molecular mechanism of this localization remains unknown. Here we identify a direct interaction between PAR-3 and KIF3A, a plus-end-directed microtubule motor protein, and show that aPKC can associate with KIF3A through its interaction with PAR-3. The expression of dominant-negative PAR-3 and KIF3A fragments that disrupt PAR-3-KIF3A binding inhibited the accumulation of PAR-3 and aPKC at the tip of the neurites and abolished neuronal polarity. These results suggest that PAR-3 is transported to the distal tip of the axon by KIF3A and that the proper localization of PAR-3 is required to establish neuronal polarity.