Eps15 homology domain-containing proteins (EHDs) are conserved ATPases implicated in membrane remodeling. Recently, EHD1 was found to be enriched at synaptic release sites, suggesting a possible involvement in the trafficking of synaptic vesicles. We have investigated the role of an EHD1/3 ortholog (l-EHD) in the lamprey giant reticulospinal synapse. l-EHD was detected by immunogold at endocytic structures adjacent to release sites. In antibody microinjection experiments, perturbation of l-EHD inhibited synaptic vesicle endocytosis and caused accumulation of clathrin-coated pits with atypical, elongated necks. The necks were covered with helix-like material containing dynamin. To test whether l-EHD directly interferes with dynamin function, we used fluid-supported bilayers as in vitro assay. We found that l-EHD strongly inhibited vesicle budding induced by dynamin in the constant presence of GTP. l-EHD also inhibited dynamin-induced membrane tubulation in the presence of GTPγS, a phenomenon linked with dynamin helix assembly. Our in vivo results demonstrate the involvement of l-EHD in clathrin/dynamin-dependent synaptic vesicle budding. Based on our in vitro observations, we suggest that l-EHD acts to limit the formation of long, unproductive dynamin helices, thereby promoting vesicle budding.