Maintaining a healthy gut environment is a prerequisite for sustainable animal production. The gut plays a key role in the digestion and absorption of nutrients and constitutes an initial organ exposed to external factors influencing the health of animals. Lactobacillus animalis (LA51) is a probiotic strain proven to improve gastrointestinal (GI) health in cattle, in part by reducing the burden of E. coli and Salmonella. Intestinal dysbiosis can promote overgrowth of theses pathogens, which in neonate cattle results in intestinal barrier damage (leaky gut) and systemic infections. The objective of this study was to evaluate in vitro beneficial effects of LA51 on GI health in the presence of pathogens. Two assays were performed. For the adhesion assay, E. coli O157 (DSM17076) was added to intestinal Caco-2 cell monolayers (5 × 10⁷ cfu/well) pre-incubated or not with LA51 (2 × 10⁸ cfu/well). E. coli adhesion was quantified by cfu enumeration using MacConkey agar plates incubated 16h at 37°C. For the “leaky gut” assay, transepithelial electrical resistance (TEER) was measured across Caco-2 monolayers exposed to LA51 (1.5 × 10⁸ cfu/transwell) with or without S. Typhimurium (DSM 19587, 0.4–1.5 × 10⁸ cfu/transwell) and incubated at 37°C, 5% CO₂. FITC-dextran (FD) was added to the apical side of the Caco-2 cells after 9h of TEER measurements. The amount of FD translocated to the basolateral side was quantified after 2h by measuring the fluorescent signal. LA51 reduced the binding of E. coli O157 to the cells by 50% (12.3 × 10⁵ cfu/mL vs. 5.9 × 10⁵ cfu/mL, P < 0.01). S. Typhimurium caused a dose-dependent TEER decrease, inversely correlated with increasing FD translocation. At each of the 3 S. Typhimurium doses tested, LA51 significantly reduced the TEER decrease (41% vs. 84% at lowest infection dose, P < 0.01) and the amount of FD translocation (0.14% vs. 0.03% at lowest infection dose, P < 0.01). In conclusion, LA51 confers protection against intestinal pathogens by reducing their adherence and by counteracting their damaging effect on the intestinal integrity.