Endocytosis and vesicle trafficking are required for optimal neural transmission. Yet, little is currently known about the evolution of neuronal proteins regulating these processes. Here, we report the first phylogenetic study of NEEP21, calcyon, and P19, a family of neuronal proteins implicated in synaptic receptor endocytosis and recycling, as well as in membrane protein trafficking in the somatodendritic and axonal compartments of differentiated neurons. Database searches identified orthologs for P19 and NEEP21 in bony fish, but not urochordate or invertebrate phyla. Calcyon orthologs were only retrieved from mammalian databases and distant relatives from teleost fish. In situ localization of the P19 zebrafish ortholog, and extant progenitor of the gene family, revealed a CNS specific expression pattern. Based on non-synonymous nucleotide substitution rates, the calcyon genes appear to be under less intense negative selective pressure. Indeed, a functional group II WW domain binding motif was detected in primate and human calcyon, but not in non-primate orthologs. Sequencing of the calcyon gene from 80 human subjects revealed a non-synonymous single nucleotide polymorphism that abrogated group II WW domain protein binding. Altogether, our data indicate the NEEP21/calcyon/P19 gene family emerged, and underwent two rounds of gene duplication relatively late in metazoan evolution (but early in vertebrate evolution at the latest). As functional studies suggest NEEP21 and calcyon play related, but distinct roles in regulating vesicle trafficking at synapses, and in neurons in general, we propose the family arose in chordates to support a more diverse range of synaptic and behavioral responses.