Actin cytoskeletal reorganization and membrane trafficking are important for spine morphogenesis. Here we investigated whether the small GTPase, ADP-ribosylation factor 6 (ARF6), which regulates actin dynamics and peripheral vesicular trafficking, is involved in the regulation of spine formation. The developmental expression pattern of ARF6 in mouse hippocampus was similar to that of the post-synaptic density protein-95, and these molecules colocalized in mouse hippocampal neurons. Overexpression of a constitutively active ARF6 mutant in cultured hippocampal neurons decreased the spine density, whereas a dominant-negative ARF6 mutant increased the density. These results demonstrate a novel function for ARF6 as a key regulator of spine formation.