Tryptophan is an essential amino acid for the human body, whose intake is through the diet. Several studies support the theory that microbiota-derived tryptophan metabolite played a crucial role in maintaining the balance between gut microbiota and the mucosal immune system. Previously, we selected the Lactobacillus plantarum KLDS 1.0386 strain with high tryptophan-metabolic activity after the screening of 16 Lactobacillus strains. The current study aimed to assess the effects of L. plantarum KLDS 1.0386 combination with tryptophan in improving ulcerative colitis (UC) induced by dextran sodium sulfate (DSS) and the potential mechanisms involved. Our results showed that L. plantarum KLDS 1.0386 combined with tryptophan (LAB + Trp) decreased DAI score, MPO level, and pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) concentration. It also increased anti-inflammatory cytokine (IL-10) production, tight junction proteins (claudin-1, occludin, and ZO-1), and mucin (MUC1 and MUC2) mRNA expressions. The level of indole-3-acetic acid (IAA), an important tryptophan metabolite in the liver, serum, and colon, was elevated after LAB + Trp treatment, which further upregulated aryl hydrocarbon receptor (AHR) mRNA expression to activate the IL-22/STAT3 signaling pathway. Moreover, the supplementation with LAB + Trp modulated gut microbiota composition. The present study provided novel insights that can be used to reduce the number of UC patients by employing a method utilizing tryptophan-catabolizing Lactobacillus strains.