Ulcerative colitis (UC) is a chronic inflammatory disease primarily affecting the colon, characterized by mucosal inflammation and ulceration. Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide derived from Entamoeba histolytica, has demonstrated the anti-inflammatory capacity. The aim of the current work was to test the protective effects of MLIF in a dextran sulfate sodium (DSS)-induced colitis mouse model. Our findings indicated that MLIF significantly inhibition of colitis development, including body weight, DAI score, colon length, and spleen index. MLIF slowing the progression of inflammation in the colon of mice exposed to DSS, evidenced by HE staining and mRNA expression levels of Il1b, Il6, Il18 and Il10. MLIF significantly alleviated intestinal barrier dysfunction in mice exposed to DSS, evidenced by AB-PAS staining and mRNA expression levels of Tjp1, Ocln and Muc2. Importantly, the administration of MLIF in colitis mice exerted beneficial effects on the gut microbiota, enhancing microbial diversity and abundance, and promoting the restoration of gut microbiota homeostasis. Non-targeted metabolomics results suggest that the benefits of MLIF may arise from its modulation of tryptophan metabolism pathways. In conclusion, MLIF prevention inflammation induction and preserves intestinal homeostasis against colitis induced by DSS.