ADP ribosylation factor 6 (Arf6) is known as a small GTPase that primarily regulates endocytic trafficking and actin cytoskeleton dynamics through the cycle of activation and inactivation by Arf-GEF and Arf-GAP, respectively. In neurons, the Arf6-signaling pathway has been shown to regulate a variety of neuronal functions, such as axonal transport, synaptic vesicle recycling, dendritic branching, spine formation, the assembly of clathrin/AP-2 complexes at synaptic membranes, and α-amino- 3-hydroxy- 5-methyl- 4-isoxazolepropionic acid-type glutamate receptor (AMPAR) internalization during long-term depression (LTD). However, the anatomical evidence of the precise localization of Arf6 in the brain has not been reported to date. In this study, we generated Arf6-specific antibody and performed multi-immunofluorescence and pre- and post-embedding immuno-electron microscopic analyses to reveal the Arf6 localization in detail in the adult mouse hippocampal neurons. Arf6 was distributed with a tiny punctate pattern in a variety of cell types, including excitatory and inhibitory neurons, astrocytes, microglia, and oligodendrocytes. In the pyramidal neurons, Arf6 showed predominant plasma membrane-associated distribution in the dendritic spines and accumulation at perisynaptic and postsynaptic density (PSD) regions. The tangential distribution pattern of Arf6 along the plasma membrane from the PSD in the spine tracks well with that of AP-2 and partly with that of clathrin. The present findings unveil a precise localization of Arf6 in the brain for the first time, serving an anatomical basis to understand the functional roles of Arf6 signaling pathway in neurons.