Growing evidence emphasizes insufficient clearance of pathological alpha-synuclein (αSYN) aggregates in the progression of Parkinson’s disease (PD). Consequently, cellular degradation pathways represent a potential therapeutic target. Prolyl oligopeptidase (PREP) is highly expressed in the brain and has been suggested to increase αSYN aggregation and negatively regulate the autophagy pathway. Inhibition of PREP with a small molecule inhibitor, KYP-2407, stimulates autophagy and reduces the oligomeric species of αSYN aggregates in PD mouse models. However, whether PREP inhibition has any effects on intracellular αSYN fibrils has not been studied before. In this study, the effect of KYP2407 on αSYN preformed fibrils (PFFs) was tested in SH-SY5Y cells and human astrocytes. Immunostaining analysis revealed that both cell types accumulated αSYN PFFs intracellularly but KYP-2047 decreased intracellular αSYN deposits only in SH-SY5Y cells, as astrocytes did not show any PREP activity. Western blot analysis confirmed the reduction of high molecular weight αSYN species in SH-SY5Y cell lysates, and secretion of αSYN from SH-SY5Y cells also decreased in the presence of KYP-2407. Accumulation of αSYN inside the SH-SY5Y cells resulted in an increase of the auto-lysosomal proteins p62 and LC3BII, as well as calpain 1 and 2, which have been shown to be associated with PD pathology. Notably, treatment with KYP-2407 significantly reduced p62 and LC3BII levels, indicating an increased autophagic flux, and calpain 1 and 2 levels returned to normal in the presence of KYP-2407. Our findings indicate that PREP inhibition can potentially be used as therapy to reduce the insoluble intracellular αSYN aggregates.