Whey protein intake is associated with modulation of energy metabolism and altered body composition both in human subjects and in animals, but the underlying mechanisms is yet not elucidated. We fed obesity prone C57BL/6J mice high-fat diets with either casein (HF Casein) or whey (HF whey) for six weeks. At equal energy intake and apparent fat- and nitrogen digestibility, mice fed HF Whey stored less energy as lipids, evident both as lower white adipose tissue mass and as reduced liver lipids, compared to HF Casein fed mice. Explorative analyses of 48 hrs urine, both by 1H-NMR and LC-MS metabolomic platforms, demonstrated higher urinary excretion of tricarboxylic acid (TCA) cycle intermediates citric acid and succinic acid (identified by both platforms), and cis-aconitic acid and isocitric acid (identified by LC-MS platform) in the HF Whey, relative to in the HF Casein fed mice. Targeted LC-MS analyses revealed higher citric acid and cis-aconitic acid concentrations in fed state plasma, but not in liver of HF Whey fed mice. We propose that enhanced urinary loss of TCA cycle metabolites drain available substrates for anabolic processes, such as lipogenesis, thereby leading to reduced lipid accretion in HF Whey fed compared to HF casein fed mice.