Significance Release of neurotransmitters relies on submillisecond coupling of synaptic vesicle fusion to the triggering signal: AP-evoked presynaptic Ca ²⁺ influx. The key player that controls exocytosis of the synaptic vesicle is the Ca ²⁺ sensor synaptotagmin 1 (Syt1). While the Ca ²⁺ activation of Syt1 has been extensively characterized, how Syt1 reversibly clamps vesicular fusion remains enigmatic. Here, using a targeted mutation combined with fluorescence imaging and electrophysiology, we show that the structural feature of Syt1 to self-oligomerize provides the molecular basis for clamping of spontaneous and asynchronous release but is not required for triggering of synchronous release. Our findings propose a mechanistic model that explains how Syt1 oligomers regulate different modes of transmitter release in neuronal synapses.