Abstract: Background: Parkinson's disease (PD) is a prevalent neurodegenerative disorder with limited therapeutic options and no cure, underscoring the urgent need for novel treatment strategies. Our previous work demonstrated that an engineered strain of Clostridium butyricum-pMTL007-GLP-1 (C. butyricum-GLP-1) alleviated PD symptoms by enhancing mitophagy, though the exact molecular mechanisms remained incompletely understood. Methods: In this study, we further investigated the neuroprotective effects and underlying mechanisms of this engineered strain using an A53T α-synuclein transgenic mouse model of PD. Specifically, we evaluated its impact on motor function, gut α-synuclein expression, intestinal barrier function, gut microbial composition, and neuropathological changes, with a focus on the PI3K/AKT/GSK-3β signaling pathway. Results: Our findings revealed that C. butyricum-GLP-1 ameliorated motor deficits in PD mice by reducing intestinal α-synuclein accumulation, restoring gut barrier function, and modulating microbial diversity—notably increasing the relative abundance of of Prevotella at the genus level. Furthermore, the engineered strain attenuated neuropathological alterations by decreasing phosphorylated α-synuclein (p-α-syn) in the substantia nigra while upregulating tyrosine hydroxylase (TH), dopamine-transporter (DAT), and glucagon-like peptide-1-receptor (GLP-1R) expression. These neuroprotective effects were associated with suppressed proinflammatory responses and enhanced anti-inflammatory and anti-apoptotic signaling, likely mediated through PI3K/AKT/GSK-3β pathway activation. Conclusions: C. butyricum-pMTL007-GLP-1 exerts significant neuroprotective effects in PD mice by reshaping gut microbiota composition and activating the PI3K/AKT/GSK-3β pathway. These findings provide further theoretical support for the potential application of probiotic-based therapies in Parkinson’s disease treatment.