Parkinson’s disease (PD) results in midbrain dopaminergic neuron degeneration and neuronal accumulation of alpha-synuclein (a-syn). The ‘brain-first vs body-first’ hypothesis of PD proposes that a-syn pathology begins in the brain then spreads to the gut, or conversely begins in the gut then spreads to the brain, via the peripheral nervous system. The gut microbiota has been implicated in PD, and early dysbiosis and a-syn accumulation in the gut have been reported in ‘body-first’ PD. However, it is unclear if these events also occur in ‘brain-first’ PD. Here we investigated whether alterations in the gut microbiota and a-syn accumulation occur in the GI tract of a ‘brain-first’ rat model of PD. Adult female rats received unilateral intranigral stereotactic injection of adeno-associated virus (AAV)-a-syn or AAV-null viral vectors. Faecal and tissue collection, microbiota profiling, HPLC, immunohistochemistry for tyrosine hydroxylase (TH), a-syn, phospho(P)a-syn, and H&E staining were conducted at 24 weeks post-surgery. AAV-a-syn significantly reduced TH-positive striatal dopaminergic terminals and striatal dopamine levels. While there were no differences in alpha and beta diversity between groups, taxonomic composition analysis found decreased abundance of several bacteria including Blautia as well as increased abundance of Streptococcus in the a-syn group. Furthermore, P-a-syn was detected in the proximal colon in the AAV-a-syn group. This study shows that intranigral a-syn resulted in gut microbiota alterations and P-a-syn accumulation in the proximal colon of a rat model of ‘brain-first’ PD. This suggests that profiling of gut microbiome alterations may be a useful early indicator of ‘brain-first’ PD.