Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrP) after conversion of the cellular prion protein (PrP). The glycosylphosphatidylinositol (GPI) anchor of PrP is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrP conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrP Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein. Here we report GPI glycan structures of human PrP isolated from human brains and from brains of a knock-in mouse model in which the mouse prion protein () gene was replaced with the human gene. LC-electrospray ionization-MS analysis of human PrP from both biological sources indicated that Gly is the ω site in PrP to which GPI is attached. Gly in human PrP does not correspond to Ser, the previously reported ω site of Syrian hamster PrP We found that ∼41% and 28% of GPI anchors in human PrPs from human and knock-in mouse brains, respectively, have -acetylneuraminic acid in the side chain. Using a sialic acid linkage-specific alkylamidation method to discriminate α2,3 linkage from α2,6 linkage, we found that -acetylneuraminic acid in PrP's GPI side chain is linked to galactose through an α2,3 linkage. In summary, we report the GPI glycan structure of human PrP, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.