Significance Posttranslational modifications (PTMs) play an important role in modulating α-syn structure, function, and pathology. Phosphorylation commonly applies to pathological α-syn aggregation in Lewy bodies and is a pathological hallmark of Parkinson’s disease (PD). Here, we obtain homogeneous α-syn fibrils formed by semisynthesized α-syn with site-specific phosphorylation at Y39. The synthetic fibril recapitulates the neuropathology and degradation resistance of endogenous pY39 α-syn aggregation in PD. Cryo-EM structure determination of the pY39 α-syn fibril reveals a fold of α-syn with the entire N terminus involved in the fibril core attracted by pY39. This structure illuminates the mechanism of pY39 pathology and highlights the importance of PTMs in defining the structure and pathology of amyloid aggregation in neurodegenerative diseases.