Cyadox is an antibiotic drug and has a potential to be used as feedstuff additives in promoting the growth of animals. However the toxicity of cyadox should be fully assessed before the application and this has prompted the current investigation on the metabolic responses of mice to cyadox exposure, using a metabonomic technique. Three groups of Kunming mice were respectively given a single dose of cyadox at three different concentrations (100 mg/kg.bw, 650 mg/kg.bw and 4000 mg/kg.bw) via gavage. We present here the metabolic alterations of urine, plasma, liver and renal medulla extracts induced by cyadox exposure. The metabolic alterations induced by cyadox exposure are dose dependent and metabolic recovery is only achieved for low and moderate levels of cyadox exposure during experimental period. Cyadox exposure resulted in a disturbance of gut microbiota, which is manifested in the depleted levels of urinary hippurate, trimethylamine-N-oxide (TMAO), dimethylamine (DMA) and trimethylamine (TMA). In addition, mice exposed to cyadox at high levels caused accumulations of amino acids and depletions of nucleotides in the liver. Furthermore, marked elevations of nucleotides and a range of organic osmolytes, such as myo-inositol, choline and glycerophosphocholine (GPC) and decreased levels of amino acids are observed in the renal medulla of cyadox exposed mice. These results suggest that cyadox exposure causes inhibition of amino acid metabolism in the liver and disturbance of gut microbiota community, influences osmolytic homeostasis and nucleic acids synthesis in both the liver and the kidney. Our work provides a comprehensive view of the toxicological effects of cyadox, which is important in animal and human food safety.