ABSTRACTDisruption of intestinal barrier homeostasis is an important pathogenic factor in conditions such as irritable bowel syndrome (IBS). Lactobacillus rhamnosus GG (LGG) improves IBS symptoms through unclear mechanisms. Previous studies utilizing colorectal adenocarcinoma cell lines showed that LGG metabolites prevented interferon gamma (IFN-gamma) induced barrier damage but the model employed limited these findings. We aimed to interrogate the protective effects of LGG on epithelial barrier function using human intestinal epithelial cultures (enteroids and colonoids) as a more physiologic model. To investigate how LGG affects epithelial barrier function, we measured FITC-Dextran (FD4) flux across the epithelium as well as tight junction zonula occludens 1 (ZO-1) and occludin (OCLN) expression. Colonoids were incubated with fecal supernatants from IBS patients (IBS-FSN) and healthy controls in the presence or absence of LGG to examine changes in gut permeability. Enteroids incubated with IFN-gamma demonstrated a downregulation of OCLN and ZO-1 expression by 67% and 50%, respectively (p<0.05). This was accompanied by increased paracellular permeability as shown by leakage of FD4. Pretreatment of enteroids with LGG prevented these changes and normalized OCLN and ZO-1 to control levels. These actions were independent of its action against apoptosis. However, these protective effects were not seen with LGG cell wall extracts, LGG DNA, or denatured (boiled) LGG. Intriguingly, IBS-FSN injected into colonoids increased paracellular permeability, which was prevented by LGG. LGG, likely due to secreted proteins, protects against epithelial barrier dysfunction. Bacterial-derived factors to modulate gut barrier function may be a treatment option in disorders such as IBS.