The kinetics of Ca²⁺ triggered synaptic vesicle fusion contains synchronous and asynchronous components. At the Calyx of Held synapse, synaptotagmin-2 has been identified as the Ca² sensor protein for synchronous vesicle release while the Ca² sensor for asynchronous vesicle release remains unknown. Sr² is a divalent cation similar in size to Ca² and often substituted for Ca² as a tool in studying asynchronous vesicle release. However, the mechanism of Sr² triggering vesicle release is still under debate. We studied the spontaneous and evoked vesicle release at the wild type and synaptotagmin-2 knock-out calyceal synapses in the existence of extracellular Sr² and found synaptotagmin-2 only mediates the Sr² triggered synchronous vesicle release but not the asynchronous and spontaneous release; the unknown divalent sensor for asynchronous vesicle release has high-affinity to Sr² and mediates the spontaneous vesicle release. Our study provided a new insight into exploring the divalent sensor in triggering asynchronous synaptic vesicle release.