The spinocerebellar ataxia type 5 (SCA5) L253P mutation in beta-III-spectrin causes high-affinity actin binding. Here we developed a CRISPR knock-in mouse to determine the in vivo impact of L253P on Purkinje neurons and motor activity, and to establish a model for future testing of SCA5 therapeutics. Significantly, the knock-in mouse shows impaired motor activity on elevated beam assays at 20 weeks. In the cerebellum, L253P causes a subcellular redistribution of beta-III-spectrin in Purkinje neurons. This is marked by loss of beta-III-spectrin in distal dendrites, accumulation of beta-III-spectrin at the plasma membrane of the soma and proximal dendrites, and formation of inclusions in the soma. The inclusions additionally contain F-actin and alpha-II-spectrin, accumulate around the nucleus, form at an early age, and are larger in homozygous beta-III-spectrinL253P/L253P compared to heterozygous beta-III-spectrinL253P/+ mice. In contrast, neurons of the hippocampus and cerebral cortex, where beta-III-spectrin is also known to be expressed, abnormally accumulate beta-III-spectrin at the plasma membrane but do not form inclusions. To gain greater insight into disease mechanisms, unbiased proteomics identified over 150 cerebellar proteins that physically associate with beta-III-spectrin. Of these, cluster analysis revealed a group of 41 proteins, including glutamate receptors, SERCA2, and CaMKII, linked to synaptic transmission. Thus, the effect of the L253P to alter beta-III-spectrin localization, including decreased levels in distal dendrites, is likely associated with a disruption of beta-III-spectrin function in postsynaptic signaling. Consistent with this, and in agreement with prior findings in knockout mice, the L253P beta-III-spectrin knock-in mouse here shows that