Cilnidipine is an antihypertensive drug that was reported to have a neuroprotective profile. The present study aimed to investigate the effect of cilnidipine on the 4-aminopyridine (4-AP)-induced glutamate release in the rat cerebral cortex using isolated nerve terminals (synaptosomes). Cilnidipine reduced the release of glutamate release induced by 4-AP in a concentration-dependent manner. This inhibitory effect was associated with a reduction in the 4-AP-induced intrasynaptosomal Ca2+ concentration elevation and was not because of an alteration of the synaptosomal membrane potential. The inhibition of glutamate release by cilnidipine was markedly reduced or eliminated in the presence of the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC and the protein kinase A inhibitor H89. The intracellular Ca2+-release inhibitors dantrolene and CGP37157, the mitogen-activated protein kinase inhibitor PD98059 or the protein kinase C inhibitor GF109203X failed to affect the action of cilnidipine. These results suggest that cilnidipine inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca2+ entry and protein kinase A activity.