Chaperone-mediated autophagy (CMA) is one of the pathways in autophagic-lysosomal protein degradation system. We have revealed that CMA is impaired by the overexpression of several proteins causing spinocerebellar ataxia (SCA) that is an autosomal dominant neurodegenerative disease characterized by cerebellar atrophy and progressive cerebellar ataxia. Therefore, we assumed that the decrease in CMA activity is related to the pathogenesis of SCA. In the present study, we investigated the effect of CMA impairment by the knockdown of LAMP2A, a lysosomal receptor for CMA substrates, in cerebellar neurons on motor function of mice. LAMP2A knockdown was conducted by the cerebellar injection of adeno-associated viral vectors, which express GFP and miRNA against LAMP2A in a neuron specific manner. LAMP2A knockdown in cerebellar neurons triggered the progressive motor dysfunction. Immunostaining of cerebellar sections revealed that GFP was mainly expressed in interneurons of molecular layer and granule cells, but not in cerebellar Purkinje cells. LAMP2A knockdown prominently decreased all types of cerebellar neurons and triggered gliosis in cerebellar cortices. These results suggest that CMA impairment in cerebellar neurons causes ataxic phenotype through the degeneration of various types of cerebellar neurons.