Parkinson’s disease (PD) is a neurodegenerative disease that actively furthers the degeneration of dopaminergic neurons in substantia nigra and is characterized by the accumulation of α-synuclein (αS) in Lewy Bodies. During oxidative stress, increased levels of free radicals induce lipid peroxidation of polyunsaturated fatty acids, leading to the generation of reactive aldehydes within the body. Specifically, 4-hydroxy-2-nonenal (HNE) is known to be a lipid peroxidation product that can post-translationally modify αS, especially histidine 50 (H50). While studies have focused on the toxic effects of HNE-modified αS oligomers, no studies have determined how the site-specific H50-HNE interaction affects the biological and physical properties of αS. The purpose of this study is to identify how the site-specific modification of H50-HNE contributes to the pathogenesis of αS. To accomplish this, αS monomers were reacted with HNE, and the site-specific modification of H50 by HNE was confirmed using trypsin digest. These adducts were used to study its effect on αS aggregation, lipid binding, and cell uptake. Congo Red assay, incorporation assay, and dynamic light scattering (DLS) experiments showed that the H50-HNE modification was enough to significantly hinder the aggregation of αS, leading to a slower aggregation kinetics and smaller aggregate sizes. Moreover, the H50-HNE modification of αS induced a two-fold increase in lipid binding affinity, which may play a significant role in neurotransmitter release or synaptic vesicle integrity. Lastly, although both αS WT and HNE-modified monomers showed low levels of uptake in SH-SY5Y cells, where they were internalized by the lysosomal/endosomal system, the accuracy of these results is unconclusive and needs be addressed in future studies. The data collectively demonstrate that H50-HNE modification alone can significantly affect the biophysical properties of αS.