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Background: One promising strategy for reducing human salmonellosis induced by Salmonella Enteritidis is to supplement animal diets with natural feed additives such as mannan oligosaccharides (MOS). Objective: The aim of this study was to investigate the potential role of Salmosan® (S-βGM), an extremely β-galactomannan-rich MOS product, in preventing epithelial barrier function disruption induced by Salmonella Enteritidis colonization in an in vitro model of intestinal Caco-2 cells in culture. Methods: Differentiated Caco-2 cells were incubated for 3 h with Salmonella Enteritidis at a multiplicity of infection 10 (MOI 10) in the absence or presence of 500 µg/mL S-βGM. Paracellular permeability (PP) was assessed by transepithelial electrical resistance (TER), D-mannitol and fluorescein isothiocyanate-dextran (FD-4) flux. Tight junction (TJ) proteins and cytoskeletal actin were also localized by confocal microscopy. Reactive oxygen species (ROS) and lipid peroxidation products were evaluated. Scanning and transmission electron microscopy were used to visualize Salmonella Enteritidis adhesion to, and invasion of, the Caco-2 cell cultures. Results: Compared to controls, TER was significantly reduced 30 %, and D-mannitol and FD-4 flux were significantly increased 374 % and 54 % in Salmonella Enteritidis-infected cultures. The presence of S-βGM in infected cultures induced total recoveries of TER and FD-4 flux to values that did not differ from control (P = 0.07 and P = 0.55, respectively), and a partial recovery of D-mannitol flux. These effects were confirmed by immunolocalization of actin, zonula occludens protein-1 (ZO-1) and occludin. Similar results were obtained for Salmonella Dublin. The protection of S-βGM on PP in infected cultures may be associated with a total recovery of ROS production to values that did not differ from control (P = 0.11). Moreover, S-βGM has the capacity to agglutinate bacteria, leading to a significant reduction in intracellular Salmonella Enteritidis of 32 % (P < 0.05). Conclusions: The results demonstrate that S-βGM contributes to protecting epithelial barrier function in a Caco-2 cell model disrupted by Salmonella Enteritidis.