Parkinson's disease (PD) is a multifaceted neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons, predominantly within the substantia nigra pars compacta (SNc) region. Dysregulation of FoxO1 has been implicated in various neurodegenerative diseases, including Alzheimer's disease and PD, yet the precise underlying mechanisms remain largely elusive. In this study, we investigated the impact of α-synuclein pre-formed fibrils (PFF) treatment on BV-2 microglial cells to elucidate alterations in molecular properties and neuronal degeneration. Surprisingly, our findings revealed that PFF treatment led to an upregulation of both FoxO1 mRNA and protein levels, and translocation to nucleus, followed by increased expression of ROS detoxification genes, such as Cat and Sod2. Moreover, PFF treatment augmented mitochondrial ROS levels, while deficiency or inhibition of FoxO1 exacerbated susceptibility to PFF-induced ROS by downregulating the expression of ROS detoxification enzymes, Cat and Sod2. Conversely, attenuation of PFF-mediated ROS induction was observed upon treatment with NAC or recombinant rSodA protein. These results underscore the significance of FoxO1 in PD pathology and its role in modulating ROS levels under PFF-induced PD conditions. Collectively, our findings offer valuable insights into the genetic mechanisms underlying PD and hold promise for the development of innovative therapeutic interventions.