O-GlcNAc modification of α-synuclein monomers leads to the formation of amyloid fibrils with a distinct core structure and reduced seeding activity in Parkinson's disease models. This study shows that O-GlcNAc modification at serine 87 (gS87) results in amyloid fibrils that are less toxic and less capable of inducing pathology in neurons and in vivo. Cryogenic electron microscopy (cryo-EM) revealed that these fibrils have a different structure compared to other amyloid fibrils. Mechanistic studies suggest that interactions with heat shock proteins (HSPs) may inhibit the seeding activity of O-GlcNAc-modified fibrils. The results indicate that O-GlcNAc modification can alter the interactome of α-synuclein fibrils, reducing their seeding activity in vivo. The study also highlights that post-translational modifications, such as O-GlcNAc, are key determinants of α-synuclein amyloid strains and pathogenicity. These findings suggest that O-GlcNAc modification may protect against the progression of neurodegenerative diseases by altering the structure and function of α-synuclein fibrils. The results demonstrate that O-GlcNAc can force the formation of an α-synuclein amyloid strain with diminished pathogenicity, providing new insights into the role of post-translational modifications in neurodegenerative diseases.O-GlcNAc modification of α-synuclein monomers leads to the formation of amyloid fibrils with a distinct core structure and reduced seeding activity in Parkinson's disease models. This study shows that O-GlcNAc modification at serine 87 (gS87) results in amyloid fibrils that are less toxic and less capable of inducing pathology in neurons and in vivo. Cryogenic electron microscopy (cryo-EM) revealed that these fibrils have a different structure compared to other amyloid fibrils. Mechanistic studies suggest that interactions with heat shock proteins (HSPs) may inhibit the seeding activity of O-GlcNAc-modified fibrils. The results indicate that O-GlcNAc modification can alter the interactome of α-synuclein fibrils, reducing their seeding activity in vivo. The study also highlights that post-translational modifications, such as O-GlcNAc, are key determinants of α-synuclein amyloid strains and pathogenicity. These findings suggest that O-GlcNAc modification may protect against the progression of neurodegenerative diseases by altering the structure and function of α-synuclein fibrils. The results demonstrate that O-GlcNAc can force the formation of an α-synuclein amyloid strain with diminished pathogenicity, providing new insights into the role of post-translational modifications in neurodegenerative diseases.