Ephexin-1 functions as guanine nucleotide exchange factors for the Rho-type GTPases which have important roles in neuronal development including axon guidance, migration, morphogenesis, and plasticity of neurons. As little is known about ephexin-1 in the cerebellum, we investigated the immunolocalization of ephexin-1 in the developing canine cerebellum. While the cellular maturation was followed by the temporal pattern, the calbindin D-28k and ephexin-1 immunoreactivities gradually increased in developing canine cerebellum. When compared to the calbindin D-28k immunoreactivities, belated ephexin-1 immunolocalization was observed in the Purkinje cells which aligned a single layer during cerebellar development. These results suggest that ephexin-1 might play an important role in the development of the Purkinje cells during the first two postnatal weeks based on its immunolocalization in the present study.