Cholinergic neurons in basal forebrain (BF) project to various brain regions including cortex and hippocampus. BF receives various inputs such as serotonergic fibers from the dorsal raphe nuclei. However, serotonin (5-HT)-induced modulatory effects on the excitatory synaptic transmission in BF are unknown. This study was aimed to elucidate 5-HT-induced modulation of glutamatergic synaptic transmission onto BF cholinergic neurons. BF cholinergic neurons were identified with Cy3-192IgG and investigated in P12-20 rat brain slices. Excitatory postsynaptic currents (EPSCs) were evoked by focal electrical stimulation. 5-HT, a 5-HT1A receptor agonist or a 5-HT1B receptor agonist inhibited the amplitude of EPSCs. In the presence of both 5-HT1A and 5-HT1B receptor antagonists, most of 5-HT-induced effect disappeared. 5-HT-induced inhibition was significantly smaller in the presence of ω-agatoxin TK than that without ω-agatoxin TK. 5-HT reduced synaptic strength by changing AMPA/ NMDA ratio. These results suggest that 5-HT inhibits glutamatergic transmission onto BF cholinergic neurons via both 5-HT1A and 5-HT1B receptors. They also suggest that 5-HT inhibits glutamatergic transmission by selectively blocking CaV2.1 (P/Q-type).