<p>In the pre-synapse, vesicle fusion is a crucial process for neurotransmitter release in the nervous system. Under physiological conditions, synaptotagmin-1 (Syt1) locks synaptic vesicles in a priming state, allowing them to undergo synchronized neurotransmitter release upon Ca²⁺ activation. Elevation of intracellular ions during diseases or injuries may lead to uncontrolled neurotransmitter release independent on Ca²⁺ influx. However, its underlying molecular mechanism remains elusive. Here we show that elevation of the intracellular Zn²⁺ concentration leads to the increased frequency of spontaneous neurotransmitter release in hippocampal neurons. In the reconstituted system with neuronal SNAREs and Syt1, Zn²⁺ markedly enhances the fusion efficiency of liposomes via its binding to the interface between tandem C2 domains of Syt1. Moreover, Syt1 exhibits an elevated capacity to bind to anionic vesicles in the context of interaction with Zn²⁺, which leads to an augmentation in vesicles docking within the synaptic active zone. Finally, the mutation of the tentative Zn²⁺-binding site (Syt1_3M) results in a loss of activation function in spontaneous release by Zn²⁺, while disruption of the primary interface and the polybasic region show negligible impact on the modulatory action of Zn²⁺. Thus, these findings suggest that with Zn²⁺, Syt1 adopts an alternative regulatory mode to drive spontaneous neurotransmitter release.</p>