Glycogen synthase kinase-3 (GSK-3), a Ser/Thr protein kinase abundantly expressed in neurons, plays diverse functions in physiological and neurodegenerative conditions. Our recent study shows that upregulation of GSK-3 suppresses long-term potentiation and presynaptic release of glutamate; however, the underlying mechanism is elusive. Here, we show that activation of GSK-3β retards the synaptic vesicle exocytosis in response to membrane depolarization. Using calcium imaging, whole-cell patch-clamp, as well as specific Ca²⁺ channel inhibitors, we demonstrate that GSK-3β phosphorylates the intracellular loop-connecting domains II and III (L_II-III) of P/Q-type Ca²⁺ channels, which leads to a decrease of intracellular Ca²⁺ rise through the P/Q-type voltage-dependent calcium channel. To further illustrate the mechanisms of GSK-3β's action, we show that activation of GSK-3β interferes with the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex through: (1) weakening the association of synaptobrevin with SNAP25 and syntaxin; (2) reducing the interactions among the phosphorylated L_II-III and synaptotagmin, SNAP25, and syntaxin; and (3) inhibiting dissociation of synaptobrevin from synaptophysin I. These results indicate that GSK-3β negatively regulates synaptic vesicle fusion events via interfering with Ca²⁺-dependent SNARE complex formation.