Shadoo (Sho) is an N-glycosylated glycophosphatidylinositol-anchored protein that is expressed in the brain and exhibits neuroprotective properties. Recently, research has shown that a reduction of Sho levels may reflect the presence of PrPSc in the brain. However, the possible mechanism by which prion infection triggers down-regulation of Sho remains unclear. In the present study, Western blot and immunohistochemical assays revealed that Sho, especially glycosylated Sho, declined markedly in the brains of five scrapie agent-infected hamsters and mice at the terminal stages. Analyses of the down-regulation of Sho levels with the emergence of PrPSc C2 proteolytic fragments did not identify close association in all tested scrapie-infected models. To further investigate the mechanism of depletion of Sho in prion disease, a Sho-expressing plasmid with HA tag was introduced into a scrapie-infected cell line, SMB-S15, and its normal cell line, SMB-PS. Western blot assay revealed dramatically decreased Sho in SMB-S15 cells, especially its glycosylated form. Proteasome inhibitor MG132 reversed the decrease of nonglycosylated Sho, but had little effect on glycosylated Sho. N-acetylglucosamine transferase inhibitor tunicamycin efficiently reduced the glycosylations of Sho and PrPC in SMB-PS cells, while two other endoplasmic reticulum stress inducers showed clear inhibition of diglycosylated PrPC, but did not change the expression level and profile of Sho. Furthermore, immunoprecipitation of HA-Sho illustrated ubiquitination of Sho in SMB-S15 cells, but not in SMB-PS cells. We propose that the depletions of Sho in scrapie-infected cell lines due to inhibition of glycosylation mediate protein destabilization and subsequently proteasome degradation after modification by ubiquitination.