Pro-inflammatory environmental factors and disruptions to the gut microbiome are recognised contributors to Parkinson’s disease, and exogenous α-synuclein fibrils spread from the gut to brain via the vagus nerve in animal models. However, the natural cause of enteric α-synuclein pathology is unknown, but emerging research implicates enteroendocrine cells in the gut epithelium. We investigated epithelial α-synuclein through confocal microscopy and western blotting of human intestinal epithelial monolayers (Aim 1), and imaging flow cytometry of murine enteroendocrine STC-1 cells (Aim 2). Aim 1 demonstrated α-synuclein staining in enteroendocrine cells and goblet cells, the latter being a novel result. Consequently, stimulation of the apical monolayer with faecal proteins revealed a non-significant trend for increased insoluble α-synuclein, suggesting modulation by gut luminal contents. To investigate the targeted effect of a microbial metabolite, STC-1 cells were exposed to lipopolysaccharide (8 and 32 ug/ml for 24 hours), with α-synuclein localisation and phosphorylation assessed through imaging flow cytometry. This enabled robust quantification of morphological and fluorescent data from large populations of cells, using objective data-processing algorithms to analyse protein expression and localisation at the single-cell level. Interestingly, 8 ug/ml LPS significantly decreased levels and altered intracellular distribution of α-synuclein in a TLR4-dependent manner, and did not cause α-synuclein aggregation or increase phosphorylated α-synuclein, such as that seen in PD. Overall, these findings demonstrate the complex interplay between gut metabolites and α-synuclein in the gut epithelium, providing insights into potential mechanisms contributing to PD.