AbstractBackgroundProgressive spreading of α‐synuclein via gut‐brain axis has been hypothesized in the pathogenesis of Parkinson's disease (PD). However, the source of seeding‐capable α‐synuclein in the gastrointestinal tract (GIT) has not been fully investigated. Additionally, the mechanism by which the GIT microbiome contributes to PD pathogenesis remains to be characterized.ObjectivesWe aimed to investigate whether blood‐derived α‐synuclein might contribute to PD pathology via a gut‐driven pathway and involve GIT microbiota.MethodsThe GIT expression of α‐synuclein and the transmission of extracellular vesicles (EVs) derived from erythrocytes/red blood cells (RBCs), with their cargo α‐synuclein, to the GIT were explored with various methods, including radioactive labeling of RBC‐EVs and direct analysis of the transfer of α‐synuclein protein. The potential role of microbiota on the EVs transmission was further investigated by administering butyrate, the short‐chain fatty acids produced by gut microbiota and studying mice with different α‐synuclein genotypes.ResultsThis study demonstrated that RBC‐EVs can effectively transport α‐synuclein to the GIT in a region‐dependent manner, along with variations closely associated with regional differences in the expression of gut‐vascular barrier markers. The investigation further revealed that the infiltration of α‐synuclein into the GIT was influenced significantly by butyrate and α‐synuclein genotypes, which may also affect the GIT microbiome directly.ConclusionBy demonstrating the transportation of α‐synuclein through RBC‐EVs to the GIT, and its potential association with gut‐vascular barrier markers and gut microbiome, this work highlights a potential mechanism by which RBC α‐synuclein may impact PD initiation and/or progression. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.