Human milk represents an optimal source of nutrition during infancy. Milk also serves as a vehicle for the transfer of growth factors, commensal microbes, and prebiotic compounds to the immature gastrointestinal tract. These immunomodulatory and prebiotic functions of milk are increasingly appreciated as critical factors in the development of the infant gut and its associated microbial community. Advances in infant formula composition have sought to recapitulate some of the prebiotic and immunomodulatory functions of milk through human milk oligosaccharide (HMO) fortification, with the aim of promoting healthy development both within the gastrointestinal tract and systemically. Our objective was to investigate the effects of feeding formulas supplemented with the HMO 2′-fucosyllactose (2′-FL) on serum metabolite levels relative to breastfed infants. A prospective, randomized, double-blinded, controlled study of infant formulas (64.3 kcal/dL) fortified with varying levels of 2′-FL and galactooligosaccharides (GOS) was conducted [0.2 g/L 2′-FL + 2.2 g/L GOS; 1.0 g/L 2′-FL + 1.4 g/L GOS]. Healthy singleton infants age 0–5 days and with birth weight > 2490 g were enrolled (n= 201). Mothers chose to either exclusively formula-feed or breastfeed their infant from birth to 4 months of age. Blood samples were drawn from a subset of infants at 6 weeks of age (n= 35–40 per group). Plasma was evaluated by global metabolic profiling and compared to a breastfed reference group (HM) and a control formula (2.4 g/L GOS). Fortification of control infant formula with the HMO 2′-FL resulted in significant increases in serum metabolites derived from microbial activity in the gastrointestinal tract. Most notably, secondary bile acid production was broadly increased in a dose-dependent manner among infants receiving 2′-FL supplemented formula relative to the control formula. 2′-FL supplementation increased secondary bile acid production to levels associated with breastfeeding. Our data indicate that supplementation of infant formula with 2′-FL supports the production of secondary microbial metabolites at levels comparable to breastfed infants. Thus, dietary supplementation of HMO may have broad implications for the function of the gut microbiome in systemic metabolism. This trial was registered at with the U.S. National library of Medicine as NCT01808105.