Nitric oxide-induced calcium release (NICR) is a novel calcium-release mechanism, originally found in the cerebellar Purkinje cells. NICR is mediated by type 1 ryanodine receptor (RyR1), an intracellular calcium release channel, and S-nitrosylation of cysteine at 3636 in RyR1 is essential for the induction of NICR. Although our previous pharmacological studies suggested involvement of NICR in cerebellar synaptic plasticity and neuronal cell death after ischemic brain injury, the physiological function of NICR has not been clarified. Thus, we generated knock-in mice (C3636A mice) expressing mutant RyR1 (C3636A RyR1) in which cysteine at 3636 was replaced with alanine, and examined possible involvement of NICR in cerebellar-dependent function. In the cerebellum of C3636A mice, NO-induced S-nitrosylation of C3636A RyR1 in the cerebellum as well as NICR in Purkinje cells were abolished. In addition, a cerebellar synaptic plasticity, long-term potentiation at parallel fiber-Purkinje cell synapse was severely impaired whereas long-term depression in the same synapse seemed to be unaffected. Furthermore, the extinction process of eyeblink conditioning was impaired in C3636A mice. These results indicate essential role of NICR in cerebellar motor learning, especially in the extinction phase.