Phosphatidylserine (PS), a negatively charged phospholipid present predominantly at the inner leaflet of the plasma membrane, has been widely implicated in many cellular processes including membrane trafficking. Along this line, PS has been demonstrated to be important for endocytosis, however, the involved mechanisms remain uncertain. By monitoring clathrin-mediated endocytosis (CME) of single vesicles in mouse chromaffin cells using cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate in the present study that the fission-pore duration is reduced by PS addition, indicating a stimulatory role of PS in regulating the dynamics of vesicle fission during CME. Furthermore, our results show that the PS-mediated effect on the fission-pore duration is Ca2+ -dependent and abolished in the absence of synaptotagmin 1 (Syt1), implying that Syt1 is necessary for the stimulatory role of PS in vesicle fission during CME. Consistently, a Syt1 mutant with a defective PS-Syt1 interaction increases the fission-pore duration. Taken together, our study suggests that PS-Syt1 interaction may be critical in regulating fission dynamics during CME.