EFA6A is a member of the guanine nucleotide exchange factors that can specifically activate ADP ribosylation factor 6 (ARF6). In this study, we identified α-actinin-1 as a possible interacting protein with EFA6A by the yeast two-hybrid screening with its C-terminal region as bait. The central region of α-actinin-1 containing a part of spectrin repeat 1 and spectrin repeats 2–3 is responsible for this interaction. In the hippocampal formation, EFA6A immunoreactivity occurred at a high level as numerous fine puncta in the strata oriens, radiatum, lacunosum-moleculare of the hippocampal CA1–3 subfields and the dentate molecular layer, whereas the immunoreactivity was faint in the neuronal cell layers and the stratum lucidum, the mossy fiber-recipient layer of the CA3 subfield. Double-immunofluorescent analyses revealed a partial overlapping of EFA6A and α-actinin at the dendritic spines of in vivo and cultured hippocampal neurons. Our present findings suggest that EFA6A may form a protein complex with α-actinin and activate ARF6 in close proximity of the actin cytoskeleton and membrane proteins in the dendritic spines.