Cerebellar outputs from the deep cerebellar nuclei (DCN) are critical for generating and controlling movement. DCN neuronal activity is primarily controlled by GABAergic inhibitory transmission by Purkinje cells in the cerebellar cortex, and is also modulated by nerve inputs originating from other brain regions within and outside the cerebellum. In this study, we examined the modulatory effects of serotonin (5-HT) on GABAergic synapses in the DCN. 5-HT decreased the amplitude of stimulation-evoked inhibitory postsynaptic currents (eIPSCs) in DCN neurons, and this effect was abolished by a 5-HT1B antagonist, SB 224289. The decrease in IPSC amplitude was associated with an increased paired-pulse ratio of the IPSC. 5-HT also decreased the frequency of miniature IPSCs without altering the amplitude. These data suggest that 5-HT presynaptically inhibited GABA release. Furthermore, 5-HT elicited a slow inward current in DCN neurons. Pharmacological studies revealed that 5-HT activated the 5-HT5 receptor, which is positively coupled to G-protein, and elicited the slow inward current through enhancement of hyperpolarization-activated cation channel activation. Finally, we examined the effects of 5-HT on the spike generation that accompanies repetitive stimulation of inhibitory synapses. 5-HT increased the spontaneous firing rate in DCN neurons due to depolarization. Increase in the 5-HT-induced tonic firing relatively decreased the contrast difference from the rebound depolarization-induced firing. However, the inhibitory transmission-induced silencing of DCN firing remained during the conditioning stimulus. These results suggest that 5-HT plays a regulatory role in spike generation and contributes to the gain control of inhibitory GABAergic synapses in DCN neurons.