The involvement of alpha-synuclein (alpha-Syn) amyloid formation in Parkinson's disease (PD) pathogenesis is supported by the discovery of alpha-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of a-Syn. Recently, the A53V mutation has been discovered, which leads to late-onset PD. In this study, we characterized for the first time the biophysical properties of AS3V, including the aggregation propensities, toxicity of aggregated species, and membrane binding capability, along with those of all familial mutations at the A53 position. Our data suggest that the AS3V mutation accelerates fibrillation of alpha-Syn without affecting the overall morphology or cytotoxicity of fibrils compared to those of the wild-type (WT) protein. The aggregation propensity for A.53 mutants is found to decrease in the following order: A53T > AS3V > WT > A53E. In addition, a time course aggregation study reveals that the AS3V mutant promotes early oligomerization similar to the case for the AS3T mutation. It promotes the largest amount of oligomer formation immediately after dissolution, which is cytotoxic. Although in the presence of membrane mimicking environments, the A53V mutation showed an extent of helix induction capacity similar to that of the WT protein, it exhibited less binding to lipid vesicles. The nuclear magnetic resonance study revealed unique chemical shift perturbations caused by the AS3V mutation compared to those caused by other mutations at the A53 site. This study might help to establish the disease-causing mechanism of A53V in PD pathology.