Significance Synaptotagmin (Syt) is the primary calcium ion (Ca ²⁺ ) sensor for regulated exocytosis. It couples Ca ²⁺ binding to soluble N -ethylmaleimide–sensitive factor attachment protein receptor-catalyzed fusion, but how this happens is unclear. Here, using a targeted mutation combined with a single-vesicle fusion optical assay, we show that the recently discovered structural feature of Syt to self-oligomerize is essential for Ca ²⁺ coupling of vesicular fusion. This suggests an elegant yet simple model in which these Syt oligomers formed at the interface of the docked vesicle physically prevent fusion until the influx of Ca ²⁺ .