CD226, a member of cell adhesion molecules, has been widely studied in the immune system; however, its expression in the CNS remains unknown. In our present study, we detected CD226 mRNA and protein in the mouse hippocampus and cerebellum by RT-PCR and Western blotting, respectively. Immunohistochemical studies found that CD226 is primarily located in the hilus of the dentate gyrus and stratum lucidum aligned along the pyramidal cells in the hippocampal CA3 area, the interspaces of granular cells and the somata of the Purkinje cells in the cerebellar cortex during adulthood. Double-staining results revealed that CD226 co-localized well with synaptic marker proteins including synaptophysin, syntaxin and PSD-95. During postnatal development, CD226 could not be detected at its adult locations until postnatal day 12; however, it was temporally expressed in the somata of neighboring or distant nuclei associated with its adult location. These results showed the diverse localization of CD226 in the mouse hippocampus and cerebellum for the first time and suggested its potential role in the CNS.