Parkinson’s disease (PD) is a prevalent neurodegenerative disease characterized by progressive motor dysfunction, and there is still no effective disease-modifying treatment. The gut microbiota residing in the gastrointestinal tract plays a critical role in the regulation of brain function. Additionally, alterations in microbial composition and reduced microbial diversity have been observed in PD. However, to the best of our knowledge, it remains unclear whether gut microbiota translocation can affect the development of PD. In the present study, an a-synuclein pre-formed fibril (PFF)-induced PD mouse model was established by intraintestinal injection, following treatment with dextran sulfate sodium (DSS). DSS exacerbated gut microbiota translocation, intestinal barrier disruption, motor dysfunction, dopaminergic neuron loss, and neuroinflammation in the PFF-induced PD mouse model through modulation of the regulatory T cell/cytotoxic T-lymphocyte-associated protein 4 (Treg/CTLA-4) axis. These findings suggest an important role of intestinal microbiota translocation in PD, highlighting the potential value of incorporating gut microbiome-targeted strategies into the treatment of this neurodegenerative disorder.