The relationship between gut microbiota and intestinal epithelial cells, particularly specialized enteroendocrine cells, such as GLP-1-secreting L-cells, is a critical aspect of gastrointestinal homeostasis and the gut-brain axis. This study delves into the molecular signalling pathways activated by the interaction between Bifidobacterium breve ncfb2258, a putative probiotic, and GLP-1-secreting L-cells. Bifidobacterium breve ncfb2258 produces conjugated linoleic acids (CLAs), which activate peroxisome proliferator-activated receptors (PPARs) and free fatty acid receptors (FFARs). This study aims to shed light on whether L-cells function as crossbarrier signal transducers and the mechanisms with which they do. Significant findings emerged through immunofluorescent labelling and calcium imaging, revealing differential expression patterns of these molecules in response to exposure to Bifidobacterium breve ncfb2258 supernatants. A significant response was observed when the human colorectal cancer cell line, NCI-H716 cells, a model of human GLP-1-secreting L-cells, were exposed to Bifidobacterium breve ncfb2258 supernatants (p = 0.003). PPARα and FFAR4 were both detected on NCI-H716 cells and while exposure to Bifidobacterium breve ncfb2258 supernatants increased the intensity of their cellular expression (p = <0.001; 0.042), expressiononly decreased with the addition of PPARα antagonist (p < 0.001) and remained elevated after the addition of FFAR4 antagonist (p = 0.999). These findings suggest the NCI-H716 cell line is suitable for exploring human GLP-1-secreting L-cells as potential cross-barrier signal transducers for the putative probiotic, B. Breve ncfb2258, and identify PPARα as potentially being involved in the molecular mechanisms of this interaction.