Background The serotonin transporter (SERT, encoded by SLC6A4) is downregulated in patients with inflammatory bowel disease (IBD) and in pre-clinical IBD models. While serotonin (5-HT) and SERT are known to play crucial roles in gut health and the microbiome, the therapeutic potential of targeting SERT in intestinal inflammation remains unexplored. Methods SERT knockout (KO) and wild-type (WT) mice were treated with dextran sulfate sodium (DSS) to induce chronic colitis. Fecal and serum samples were subjected to metabolomic analysis. Bioinformatic tools identified enriched metabolic pathways and microbial-related metabolites. Disease severity was assessed via myeloperoxidase (MPO) activity, RT-PCR, immunoblotting, and immunohistochemistry. Tight junction integrity was evaluated using immunofluorescence, Western blotting, RT-PCR, and serum endotoxin levels. Spearman correlation coefficients were calculated to determine the relationship between metabolite abundance and disease activity. Results SERT KO mice with chronic colitis exhibited increased disease severity. Baseline metabolomic profiles of SERT KO mice resembled those of WT mice with chronic colitis, indicating a predisposition to metabolic dysregulation. Metabolomic analysis revealed distinct fecal and serum profiles associated with chronic colitis, mirroring human IBD, including variations in purine and plasmalogen metabolites. SERT deficiency alone led to altered tryptophan metabolism, oxidative metabolism changes, and dysregulation of phosphatidylcholines and plasmalogens. Mechanistically, SERT deficiency in an inflammatory context resulted in reduced ileal tight junction expression and increased serum endotoxin levels, exacerbating disease. Additionally, several microbial metabolites in feces and serum correlated with disease activity. Specifically, indolepropionate levels were reduced in the serum of both chronic colitis and SERT KO mice, with significant negative correlations with histology scores and MPO activity. Conversely, tyrosol excretion was increased in feces of both chronic colitis and SERT KO mice, showing significant positive correlations with histology scores and MPO. Conclusion SERT deficiency exacerbates DSS-induced chronic inflammation, disrupts intestinal barrier function, and causes microbially influenced metabolic changes corresponding to disease activity. This study highlights novel changes in host-derived and bacteria-derived metabolites in serum and feces due to SERT loss. Our findings suggest that SERT deficiency leads to a loss of beneficial anti-inflammatory and antioxidant compounds. Future research will explore the therapeutic potential of fecal supernatants containing these beneficial compounds for treating intestinal inflammation. These findings underscore the disruptive role of SERT in metabolic pathways associated with IBD and suggest potential therapeutic targets for intestinal inflammation.