Low threshold voltage activated Kv1 potassium channels play key roles in regulating action potential (AP) threshold, neural excitability, and synaptic transmission. Kv1 channels are highly expressed in the cerebellum and mutations of human Kv1 genes are associated to episodic forms of ataxia (EAT-1). Besides the well-established role of Kv1 channels in controlling the cerebellar basket-Purkinje cells synapses, Kv1 channels are expressed by the deep cerebellar nuclear neurons (DCNs) where they regulate the activity of principal DCNs carrying the cerebellar output. DCNs include as well GABAergic neurons serving important functions, such as those forming the inhibitory nucleo-olivary pathway, the nucleo-cortical DCNs providing feed-back inhibition to the cerebellar cortex, and those targeting principal DCNs, but whether their function is regulated by Kv1 channels remains unclear. Here, using cerebellar slices from mature GAD67-GFP mice to identify putative GABAergic-DCNs (GAD + DCN) we show that specific Kv1 channel blockers (dendrotoxin-alpha/I/K, DTXs) hyperpolarized the threshold of somatic action potentials, increased the spontaneous firing rate and hampered evoked high frequency repetitive responses of GAD + DCNs. Moreover, DTXs induced somatic depolarization and tonic firing in previously silent, putative nucleo-cortical DCNs. These results reveal a novel role of Kv1 channels in regulating GABAergic-DCNs activity and thereby, cerebellar function at multiple levels.