Our previous studies have shown that Lactiplantibacillus plantarum DPUL-S164-derived indole-3-lactic acid (ILA) ameliorates intestinal epithelial cell barrier injury by activating aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2related factor 2 (Nrf2) signaling pathways and promoting tight junction protein expression. This study further explored the crucial substances of L. plantarum DPUL-S164 in alleviating intestinal barrier damage in mice through a dextran sodium sulfate-induced ulcerative colitis mouse model. Compared to dead L. plantarum DPUL-S164 (D-S164), live L. plantarum DPUL-S164 (S164) and its tryptophan metabolite, ILA, showed an effective ameliorating effect on the intestinal barrier injury of mice treated by antibiotic cocktail and sodium dextran sulfate, suggesting that the crucial substances of L. plantarum DPUL-S164 ameliorating intestinal barrier injury are its extracellular metabolites. Furthermore, S164 and its tryptophan metabolite, ILA, ameliorate intestinal barrier injury and suppress intestinal inflammation by activating the AhR−Nrf2 pathway and inhibiting the nuclear factor kappa-B (NF-κB) pathway. These results suggest that L. plantarum DPUL-S164 ameliorates intestinal epithelial barrier damage in mice, primarily by producing ILA as a ligand to activate the AhR pathway.