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 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.