The mechanisms that regulate synaptic vesicle recycling are unclear. Here, Li et al. show that Ca2+ sensors that drive vesicle fusion also influence vesicle endocytosis. High-resolution recordings of fluorescently tagged vesicles revealed a role of synaptotagmin 1 in the regulation of the endocytosis of single synaptic vesicles. Furthermore, whereas synaptotagmin 1 and other canonical vesicle-fusion machinery components facilitate rapid multivesicular endocytosis after repetitive stimulation, synaptotagmin 7 promotes asynchronous neurotransmitter release and targets vesicles towards a slow endocytic pathway.