Spinocerebellar ataxia type 3 (SCA3) is a human neurodegenerative disorder caused by the expansion of CAG repeats in the coding region of the ataxin-3 gene. We generated the first humanized SCA3 knock-in mouse model by introducing human cDNA for ataxin-3 with 91 CAG repeats into the mouse ataxin-3 locus. The resulting animals express human mutant ataxin-3 protein in multiple brain structures and non-neuronal tissues. Like in human patients, the humanized allele shows both somatic and intergenerational CAG instability. The intergenerational instability is significantly associated with the gender of parent. Offspring inherits expanded CAG repeats in paternal transmissions and contracted CAG repeats in maternal transmissions. Moreover, mice show early upregulation of Serpina3n gene expression in the brain as early as at 7 weeks of age. This upregulation is also present in astrocytes isolated from neonatal animals, which suggest that mutant ataxin-3 has a more direct influence on a Serpina3n expression. The knock-in animals also demonstrate histopathological hallmarks of SCA3, including the damage of Purkinje cells in the cerebellum and the presence of intranuclear ataxin-3 inclusions.