Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract and is strongly associated with intestinal immunity and the microbiome. Tryptophan (Trp) is an inflammatory inhibitor and modulator of the intestinal microflora. We explored the serum profile of amino acids and the effects of diet supplementation with Trp (1.0 g kg-1) on intestinal immunity and microbiota in the acetic acid-induced colitis mouse model. We analyzed the survival rate, colonic morphological parameters, profiles of serum amino acids, microbiota in colonic contents and the relative gene abundance of intestinal proinflammatory cytokines. Although the dietary Trp supplementation failed to improve the survival rate and ameliorate the morphological parameters of colon in mice with colitis, Trp modulated the general serum amino acid profile by reducing the amino acid profiles of threonine, methionine and proline, affected intestinal immunity by inhibiting the colonic expression of interleukin-22 and changed the microbiota by reducing the abundance of Candidatus, Clostridium and Coprococcus at the genus level. In conclusion, dietary Trp supplementation in a mouse model of colitis did not ameliorate the survival rate and morphological parameters of colon but did modulate the serum amino acid profiles, intestinal immunity and microbiota. These findings enhance our understanding of the roles of Trp in the metabolism of serum amino acids, intestinal immunity and microbiota.