Listeria monocytogenes is a foodborne pathogen of particular concern to neonates and the immunocompromised. During initial infection, L. monocytogenes invades intestinal epithelial cells by entry into a phagosome followed by phagosome escape, cytosolic replication, and dissemination to neighboring cells. Vacuolar escape is followed by modulation of host signaling cascades leading to endoplasmic reticulum stress and activation of the unfolded protein response. Prebiotic oligosaccharides have been shown to minimize infections by gut pathogens in live stock and humans. Mannanoligosaccharides such as Biomos are used as a preventative against GI pathogen colonization while human milk oligosaccharides (HMO) are thought to confer protection to neonates against pathogen infection. Prebiotic oligosaccharides work either by acting as a decoy host receptor for pathogen binding, or by modulating the host cell membrane composition to reduce pathogen association. The mechanism of action for Biomos and HMO are currently unknown.Efficacies of Biomos and HMO in blocking pathogen association with differentiated colonic epithelial cells (Caco-2) were evaluated using a modified gentamicin protection assay in the presence of each oligosaccharide. Host and pathogen transcriptomes were determined using RNAseq.In this study, we demonstrate that Biomos increases L. monocytogenes host (Caco-2) association while HMO decreases Caco-2 association. Analysis of the Caco-2 transcriptome during infection with prebiotic treatment revealed repression of the ER stress response and inflammatory cascades. Our results suggest that L. monocytogenes gain access to the cytosol with Biomos treatment but remain vacuole-associated with HMO treatment. Although cytosolic L. monocytogenes induces STING expression through c-di-AMP secretion, the ER stress signaling cascade is not activated due to repression of the STING binding partner, TBK1. Taken together, these results suggest that Biomos and HMO modulate L. monocytogenes host association via different mechanisms of action and result in differential subcellular localization during host invasion.