The role of Ca²⁺ in synaptic vesicle endocytosis remains uncertain due to the diversity in various preparations where several forms of endocytosis may contribute variably in different conditions. Although recent studies have demonstrated that Ca²⁺ is important for clathrin-mediated endocytosis (CME), the mechanistic role of Ca²⁺ in CME remains to be elucidated. By monitoring CME of single vesicles in mouse chromaffin cells with cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate that the dynamics of vesicle fission during CME is Ca²⁺ dependent but becomes Ca²⁺ independent in synaptotagmin 1 (Syt1) knock-out cells. Our results thus suggest that Syt1 is necessary for the Ca²⁺ dependence of CME.