Deoxynivalenol (DON), a prevalent mycotoxin contaminating cereal crops globally, poses significant threats to animal and human health through its gastrointestinal toxicity. While DON-induced intestinal damage has been documented in mammals, its metabolic mechanisms in aquatic species remain poorly understood, particularly in adult zebrafish models that offer unique advantages for toxicological studies. Multi-omics analysis revealed 16 key differential metabolites (9 upregulated, 7 downregulated) associated with amino acid metabolism and carbohydrate homeostasis. Pathway enrichment analysis identified significant perturbations in 2-oxocarboxylic acid metabolism and sphingolipid signaling, suggesting mitochondrial dysfunction and epithelial barrier disruption as primary toxicity mechanisms. This study establishes the first adult zebrafish model for DON intestinal toxicity evaluation, demonstrating its utility in revealing conserved metabolic targets across species. The identified pathway-specific biomarkers provide novel insights for developing dietary interventions against mycotoxin exposure.