Emerging evidence suggests that gastrointestinal dysfunction and enteric nervous system pathology play a critical role in the early stages of Parkinson's disease. Considering the bidirectional relationship between gastrointestinal symptoms and mood disorders, this study aimed to elucidate the effects and possible mechanisms of action of vortioxetine, a serotonergic antidepressant, on the pathophysiological changes induced by rotenone in the enteroglial cells. α-synuclein, phosphorylated α-synuclein, TLR2, S100B and RAGE expression were detected in duodenal tissues of rats administered rotenone (2 mg/kg/day, s.c.) and/or vortioxetine (10 mg/kg/day, s.c.) for 28 days. For the mechanism of action studies, rat-derived enteroglial cells were treated with rotenone (10 μM) and/or vortioxetine (5 μM or 1 μM) for 24 h. The effects of vortioxetine were evaluated in the presence of the TLR2 antagonist C29, RAGE antagonist FPS-ZM1 and the S100B inhibitor pentamidine. TLR2, S100B, RAGE, and NFκB mRNA levels and proinflammatory cytokines via RT-qPCR and ELISA. Our results demonstrate that rotenone treatment significantly increased α-synuclein, pS129-α-synuclein, TLR2, and S100B expression while reducing RAGE levels, indicating marked enteric pathology. Vortioxetine administration attenuated these effects, reducing α-synuclein accumulation and proinflammatory markers. In vitro, rotenone impaired glial responses, decreasing S100B, RAGE, and NFκB markers, while vortioxetine improved these responses, promoting resynthesis of inflammatory molecules. Notably, S100B, NFκB, and cytokine levels (TNF-α, IL-1β, IL-6) were affected by C29, FPS-ZM1, and pentamidine pretreatments. Thus, vortioxetine is thought to have beneficial effects on rotenone-induced pathological changes in EGCs, and some of these effects are thought to be mediated by the TLR2/S100B/RAGE pathway.