The effect of cycloheterophyllin, a prenylflavone isolated from Artocarpus heteophyllus, on glutamate release was studied in the rat hippocampus using synaptosome and slice preparations. In rat hippocampal synaptosomes, cycloheterophyllin inhibited 4-aminopyridine (4-AP)-evoked glutamate release and elevation of intrasynaptosomal calcium levels. The inhibitory effect of cycloheterophyllin on 4-AP-evoked glutamate release was prevented in the presence of the vesicular transporter inhibitor, the N- and P/Q-type calcium channel blocker, and the protein kinase C (PKC) inhibitor but was insensitive to the intracellular Ca2+ release inhibitors, the protein kinase A inhibitor, and the mitogen-activated/extracellular signal-regulated kinase inhibitor. Western blotting data in synaptosomes also showed that cycloheterophyllin significantly decreased the level of phosphorylation of PKC. In addition, cycloheterophyllin decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) without influencing the amplitude of sEPSCs and glutamate-activated currents in hippocampal slices, supporting a presynaptic action. Together, these results suggest that cycloheterophyllin inhibits presynaptic glutamate release by suppressing N- and P/Q-type calcium channel and PKC activity in the rat hippocampus.