Parkinson's disease (PD) is the second most common neurodegenerative disease, in which mitochondrial dysfunction and abnormal aggregation of alpha-synuclein (α-syn) play key roles in the pathology of PD. As a classic tumor suppressor, p53 has also been found to be involved in the pathological process of PD in recent years. However, the specific mechanism by which p53 regulates mitochondrial function and abnormal aggregation of α-syn is still unclear. Here, we observed that the expression of α-syn and p53 was increased and mitochondria were impaired in the MPTP-induced PD mouse model, leading us to propose speculation on whether p53 affects mitochondrial impairment and abnormal α-syn aggregation in PD pathology. Next, cellular experiments revealed that the p53 inhibition by pifithrin-α regulates mitochondrial function through mitophagy and mitochondrial dynamics, then ameliorates oxidative stress and apoptosis in PD. Meanwhile, the in vitro study showed that the p53 protein interacted with α-syn to accelerate the process of α-syn liquid-liquid phase separation and amyloid fibril formation, promoting the development of PD pathology. In summary, p53 modulates mitochondrial function through mitophagy and mitochondrial dynamics, stimulating the pathogenic aggregation of α-syn protein and neurodegeneration in Parkinson's disease.