The aim of this paper was to study the effect of combination of Lactobacillus strains and tryptophan (Trp)-rich diet on the intestinal barrier function of Balb/c mice exposed to a cocktail of antibiotics and dextran sodium sulfate. Several Lactobacillus strains isolated from the healthy human fecal sample was found to utilize Trp to produce indole derivatives. The results of Trp metabolism indicated that the ability of Lactobacillus to metabolize Trp to produce indole-3-lactic acid (ILA), indole-3-carboxaldehyde (I3C), and indole-3-acetic acid varies in vitro and in vivo. The effect of Lactobacillus with high-yielding indole derivatives on disease activity index, colon length, and intestinal permeability was significantly better than that of Lactobacillus with low-yielding indole derivatives in a high Trp diet. And Lactobacillus combined with Trp intervention also had a certain regulatory effect on the intestinal flora of male BALB/c mice. Among them, Lactiplantibacillus plantarum DPUL-S164 produced more ILA both in vivo and in vitro, and the combination of L. plantarum DPUL-S164 and Trp significantly decreased the expression level of the serum pro-inflammatory cytokine interleukin (IL)-6 and increased the expression level of the anti-inflammatory cytokine IL-10, significantly improved the number of goblet cells in the mouse mucous layer and increased mucin and tight junction protein expression. Furthermore, L. plantarum DPUL-S164 combined with Trp intervention activated the aryl hydrocarbon receptors (AhR) signaling pathway. Furthermore, we found that the expression of colonic tight junction protein was positively correlated with the expression of colonic AhR, and the expression of AhR was positively correlated with the concentrations of ILA and I3C in vivo. Therefore, we conclude that the ILA as AhR ligand produced by L. plantarum DPUL-S164 regulated the AhR pathway, thus up-regulating the expression of the tight junction protein and protecting the integrity of the epithelial barrier.