A low-affinity Ca2+/H+-antiport was described in the membrane of mammalian brain synaptic vesicles. Electrophysiological studies showed that this antiport contributes to the extreme brevity of excitation-release coupling in rapid synapses. Synaptotagmin-1, a vesicular protein interacting with membranes upon low-affinity Ca2+-binding, plays a major role in excitation-release coupling, by synchronizing calcium entry with fast neurotransmitter release. Here, we report that synaptotagmin-1 is necessary for expression of the vesicular Ca2+/H+-antiport. We measured Ca2+/H+-antiport activity in vesicles and granules of pheochromocytoma PC12 cells by three methods: (i) Ca2+-induced dissipation of the vesicular H+-gradient; (ii) bafilomycin-sensitive calcium accumulation and (iii) pH-jump-induced calcium accumulation. The results were congruent and highly significant: Ca2+/H+-antiport activity is detectable only in acidic organelles expressing functional synaptotagmin–1. In contrast, synaptotagmin-1-deficient cells – and cells where transgenically encoded synaptotagmin-1 was acutely photo-inactivated – were devoid of any Ca2+/H+-antiport activity. Therefore, in addition to its previously described functions, synaptotagmin-1 is involved in a rapid vesicular Ca2+ sequestration through a Ca2+/H+ antiport.