Synaptic vesicle recycling is essential for maintaining normal synaptic function. The coupling of exocytosis and endocytosis is assumed to be Ca2+ dependent, but the exact role of Ca2+ and its key effector synaptotagmin-1 (syt1) in regulation of endocytosis is poorly understood. Here, we probed the role of syt1 in single- as well as multi-vesicle endocytic events using high-resolution optical recordings. Our experiments showed that the slowed endocytosis phenotype previously reported after syt1 loss of function can also be triggered by other manipulations that promote asynchronous release such as Sr2+ substitution and complexin loss of function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles toward slow retrieval by the asynchronous release Ca2+ sensor synaptotagmin-7. In contrast, after single synaptic vesicle fusion, syt1 acted as an essential determinant of synaptic vesicle endocytosis time course by delaying the kinetics of vesicle retrieval in response to increasing Ca2+ levels. •Loss of syt1, complexins, or Sr2+ substitution slows endocytosis after activity•Asynchronously released synaptic vesicles are retrieved via slow endocytosis•Link between asynchronous release and slowed endocytosis requires synaptotagmin-7•Whereas, after synchronous single synaptic vesicle fusion, syt1 delays endocytosis In this study, Li and colleagues demonstrate that synaptic vesicle fusion machinery, comprised of synaptotagmin-1, complexins, and synaptotagmin-7, in addition to determining the timing and Ca2+ dependence of neurotransmitter release, also dictates the properties of subsequent synaptic vesicle endocytosis.