Synaptic vesicle fusion at synapses is triggered by increases in cytosolic Ca²⁺ levels. However, the identity of the Ca²⁺ sensor and the transduction mechanism of the Ca²⁺ trigger are unknown. We show that Complexins, stoichiometric components of the exocytotic core complex, are important regulators of transmitter release at a step immediately preceding vesicle fusion. Neurons lacking Complexins show a dramatically reduced transmitter release efficiency due to decreased Ca²⁺ sensitivity of the synaptic secretion process. Analyses of mutant neurons demonstrate that Complexins are acting at or following the Ca²⁺-triggering step of fast synchronous transmitter release by regulating the exocytotic Ca²⁺ sensor, its interaction with the core complex fusion machinery, or the efficiency of the fusion apparatus itself.