Formula-fed (FF) infants exhibit a different metabolic profile than breast-fed (BF) infants. Two potential mechanisms are the higher protein level in formula compared with breast milk and the removal of the milk fat and associated milk fat globule membranes (MFGM) during production of infant formula. To determine whether MFGM may impact metabolism, formula-fed infants were randomly assigned to receive either an MFGM isolate-supplemented experimental formula (EF) or a standard formula (SF) from 2 until 6 months and compared with a BF reference group. Infants consuming EF had higher levels of fatty acid oxidation products compared to infants consuming SF. Although the protein level in the study formula was approximately 12 g/L (lower than most commercial formulas), a metabolic difference between FF and BF remained such that FF infants had higher levels of amino acid catabolism by-products and a low efficiency of amino acid clearance (preference for protein metabolism). BF infants had higher levels of fatty acid oxidation products (preference for fat metabolism). These unique, energy substrate-driven metabolic outcomes did not persist after diet was shifted to weaning foods and appeared to be disrupted by complementary feeding. Our results suggest that MFGM may have a role in directing infant metabolism.