Synaptotagmin I is a synaptic vesicle protein that is thought to act as a Ca²⁺ sensor in neurotransmitter release. The first C₂ domain of synaptotagmin I (C₂A domain) contains a bipartite Ca²⁺-binding motif and interacts in a Ca²⁺-dependent manner with syntaxin, a central component of the membrane fusion complex. Analysis by nuclear magnetic resonance spectroscopy and site-directed mutagenesis shows that this interaction is mediated by the cooperative action of basic residues surrounding the Ca²⁺-binding sites of the C₂A domain and is driven by a change in the electrostatic potential of the C₂A domain induced by Ca²⁺ binding. A model is proposed whereby synaptotagmin acts as an electrostatic switch in Ca²⁺-triggered synaptic vesicle exocytosis, promoting a structural rearrangement in the fusion machinery that is effected by its interaction with syntaxin.