BACKGROUND AND AIMS: Although the anti-inflammatory benefits of exercise are well-documented, the specific mechanisms responsible for these advantages remain uncertain. N-lactoyl-phenylalanine (Lac-Phe), a major metabolite produced during exercise, is synthesized through the condensation of lactic acid and phenylalanine, catalyzed by the CNDP2. However, the potential anti-inflammatory properties of Lac-Phe remain poorly understood. This study aimed to investigate the anti-inflammatory effects of Lac-Phe in the context of inflammatory bowel disease (IBD) and to examine the underlying mechanisms. METHODS: The levels of Lac-Phe were measured in both IBD patients and mice utilizing ELISA kits. The anti-inflammatory effects of Lac-Phe were demonstrated through colitis models. The impacts of Lac-Phe on macrophage polarization and the associated mechanisms were determined by flow cytometry, qPCR, RNA sequencing, Western blotting, and immunofluorescence. RESULTS: Our study revealed a reduction in plasma Lac-Phe content in IBD patients, in conjunction with a decrease in the expression of CNDP2 in the colon, which exhibited a negative correlation with disease activity scores. Exercise mitigated DSS-induced colitis in mice by elevating plasma Lac-Phe levels and inhibiting the polarization of M1 macrophages. Mechanistically, Lac-Phe impedes the movement of p65 protein from the cytoplasm into the nucleus, consequently suppressing the activation of the NF-kappaB signaling pathway and macrophage M1 polarization. Furthermore, the supplementation of phenylalanine, a substrate of Lac-Phe, was observed to enhance the generation of Lac-Phe and to exert a protective effect in the murine colitis model. CONCLUSION: Our results suggest that exercise can induce the production of Lac-Phe, which plays a preventive role against DSS-induced colitis in mice. Lac-Phe mitigates colitis through inhibition of the polarization of M1 macrophage. Adjusting macrophage polarization with Lac-Phe and phenylalanine supplementation may offer a potential therapeutic strategy for managing IBD.