Recently we showed that exchanging intact casein with extensively hydrolysed casein in Western diets prevented diet-induced obesity in obesity-prone C57BL/6J mice. To gain further insight into the underlying mechanisms for the metabolic alterations induced by intake of hydrolysed casein, we performed an exploratory investigation using proton NMR spectroscopy, multi-block PCA (MBPCA) and a multi-compartment model including analyses of plasma, urine, faeces and tissue samples from mice fed diets with intact or hydrolysed casein and 16 or 32 energy% protein. The MBPCA superscores showed a clear separation between samples from mice fed intact and hydrolysed casein diets, respectively. Block loadings revealed that fecal fat content was higher, and tissue and plasma lipid levels were lower in mice fed hydrolysed casein diets compared with mice fed intact casein. Amino acid metabolism was also altered by dietary protein form, and levels of branched-chain amino acids were higher in faeces and urine and lower in plasma and spleen in mice fed hydrolysed protein. Moreover, hepatic levels of the sulphur-containing metabolites taurine and glutathione were increased in mice fed hydrolysed casein, and hepatic glycogen amount was increased in mice fed hydrolysed casein. In contrast, the levels of glucose and its metabolite lactate were reduced in faeces, liver and plasma. Taken together, NMR-based metabolomic analyses indicated that pathways within lipid, amino acid and carbohydrate metabolism were altered by intake of hydrolysed casein, and that these alterations are likely to be underlying mechanisms for the observed prevention against diet-induced obesity associated with hydrolysed casein intake.