Simple Summary: The intestinal epithelial barrier is a critical host defense mechanism and plays an important role in the gastrointestinal health of animals. Impaired gut barrier integrity caused by enteric pathogenic infections or ingested mycotoxins is a particular concern in farm animals, as they may trigger intestinal inflammation and possibly chronic pathological conditions. Ligilactobacillus animalis (formerly known as Lactobacillus animalis) 506 is a probiotic lactic acid-producing bacterial strain used in commercial products to support the health and performance of beef and dairy cattle. However, limited information is available regarding the mechanism underlying its proposed in vivo benefits. This study investigated the ability of L. animalis 506 to support gut barrier integrity and inflammation in vitro. The results showed that L. animalis 506 supported gut barrier integrity and regulated the release of interleukin (IL)-8 from epithelial cells upon challenge with enteric pathogenic isolates. Moreover, L. animalis 506 mitigated epithelial barrier disruption and tight junction protein redistribution caused by the frequent food contaminant mycotoxin deoxynivalenol (DON). These results provide new insights into the potential mechanisms by which L. animalis 506 might benefit farm animals. This study investigated the impact of L. animalis 506 on gut barrier integrity and regulation of inflammation in vitro using intestinal epithelial cell lines. Caco-2 or HT29 cell monolayers were challenged with enterotoxigenic E. coli (ETEC) or a ruminant isolate of Salmonella Heidelberg in the presence or absence of one of six probiotic Lactobacillus spp. strains. Among these, L. animalis 506 excelled at exerting protective effects by significantly mitigating the decreased transepithelial electrical resistance (TEER) as assessed using area under the curve (AUC) (p < 0.0001) and increased apical-to-basolateral fluorescein isothiocyanate (FITC) dextran translocation (p < 0.0001) across Caco-2 cell monolayers caused by S. Heidelberg or ETEC, respectively. Similarly, L. animalis 506 and other probiotic strains significantly attenuated the S. Heidelberg- and ETEC-induced increase in IL-8 from HT29 cells (p < 0.0001). Moreover, L. animalis 506 significantly counteracted the TEER decrease (p < 0.0001) and FITC dextran translocation (p < 0.0001) upon challenge with Clostridium perfringens. Finally, L. animalis 506 significantly attenuated DON-induced TEER decrease (p < 0.01) and FITC dextran translocation (p < 0.05) and mitigated occludin and zona occludens (ZO)-1 redistribution in Caco-2 cells caused by the mycotoxin. Collectively, these results demonstrate the ability of L. animalis 506 to confer protective effects on the intestinal epithelium in vitro upon challenge with enteric pathogens and DON known to be of particular concern in farm animals. [ABSTRACT FROM AUTHOR]