α-Synuclein, the major aggregating protein in Parkinson's disease, can be modified by the small protein SUMO, indicating a potential role in disease. However, the effects of SUMOylation on α-synuclein aggregation remain controversial due to heterogeneous nature of the proteins previously investigated. Here we used protein semisynthesis to obtain homogeneously SUMOylated α-synuclein and discovered site- and isoform-dependent effects of SUMOylation on α-synuclein aggregation. Our results indicate that SUMOylation at K102 is a better inhibitor of aggregation than corresponding modification at K96 and SUMO1 modification, a better inhibitor than SUMO3.