The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF5 is a heterotetrameric motor that conveys vesicles and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein(s) that interacts with the tail region of KIF5 and found a specific interaction with βIII spectrin. The amino acid residues between 1394 and 1774 of βIII spectrin were required for the interaction with KIF5C. βIII spectrin also bound to the tail region of neuronal KIF5A and ubiquitous KIF5B but not to other kinesin family members in the yeast two-hybrid assay. In addition, these proteins showed specific interactions, confirmed by GST pull-down assay and co-immunoprecipitation. βIII spectrin interacted with GST-KIF5 fusion proteins, but not with GST alone. An antibody to βIII spectrin specifically co-immunoprecipitated KIF5s associated with βIII spectrin from mouse brain extracts. These results suggest that KIF5 motor proteins transport vesicles or organelles that are coated with βIII spectrin.