A growing body of evidence suggests that microbial tryptophan metabolites play a crucial role in maintaining intestinal barrier stability and modulating host immunity. Our previous study showed that the ( ) DPUL-S164 intervention in mice with a high tryptophan (Trp) diet promotes indole-3-lactic acid (ILA) production in the mice's intestinal tract and ameliorates dextran sodium sulfate(DSS)-induced intestinal barrier damage in mice. In this study, we used the HT-29 cell monolayer model to evaluate the effect of the DPUL-S164 Trp metabolites (DPUL-S164-TM) on the intestinal barrier. We found that DPUL-S164-TM alleviated lipopolysaccharide (LPS)-induced intestinal barrier damage and inflammation of the HT-29 cell monolayer by promoting the expression of tight junction proteins (ZO-1, occludin, claudin1), activating the AhR and Nrf2 signaling pathways, and inhibiting the NF-κB signaling pathway. We found that the promotion of tight junction protein expression and the activation of the Nrf2 signaling pathway by DPUL-S164-TM were dependent on the AhR expression of HT-29 cells. Additionally, DPUL-S164-TM showed a dramatic increase in the ILA content. Therefore, we inferred that ILA in DPUL-S164-TM plays a key role in improving the intestinal barrier function and alleviating inflammation.