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A metabonomics approach based on high resolution 1H NMR spectroscopy was applied to investigate the metabolite fingerprints in urine and serum samples from db/db mice of 8 weeks old, an animal model of type 2 diabetes mellitus (T2DM). Both NMR spectra and metabonomics results were discussed and the variations on related metabolic pathway were analyzed. The urinary excretions of diabetic mice have elevated levels of citrate, alanine, acetate, TMAO, hippurate, taurine, creatinine, succinate, pyruvate, glycine in addition to evident increase of glucose compared to the control ones. The metabolic variation in serum samples of db/db mice is marked by the increases of lactate, 3-hydroxybutyrate, glutamine, glutamate and choline and the decreases of leucine and valine. These results indicate that NMR-based metabonomics is an efficient approach for investigating the subtle metabolic alterations in urine and serum from diabetic mice and the findings of the characteristic metabolites would be helpful for early diagnosis and prevention of T2DM and its related complications.