Antibiotics, including enrofloxacin (ENR), are widely used for bacterial disease treatment in aquaculture in China, resulting in their discharge into various aquatic environments. Therefore, researchers have focused their attention on the harmful effects of antibiotics on aquatic animal metabolism. To understand the impacts of ENR on the metabolism of cultured American shad, the guts of these fish were sampled to test the toxicity of ENR through the combined results of metabolomics and transcriptomics analyses. In this study, adult American shad were exposed to ENR (200 mg/kg) for 30 days. The results showed that ENR could significantly (p< 0.05) increase the body weight of American shad. ENR exposure also contributed to the dysregulation of intestinal metabolism (approximately 49 metabolites and 963 genes). Multiomics analyses of the responses indicated that the specific metabolic dysregulation caused by ENR affects many signaling pathways, such as the glycolysis/gluconeogenesis and pentose phosphate pathways, which are known to be linked with body weight gain through protein and lipid accumulation. In conclusion, ENR treatment affected the metabolic system in the gut of American shad. The transcriptomics and metabolomics analysis results provide essential data for evaluating antibiotic hazards in the guts of aquatic organisms.