Background: Human milk contains high concentrations and diversity of sialylated oligosaccharides that have multifunctional health benefits, however, their potential role in optimizing neurodevelopment remains unknown.Objective: To investigate the effect of sialylated milk oligosaccharides (SMOS) intervention on neurotransmitters and brain metabolites in piglets.Methods: 3-day-old piglets were randomly allocated to one of three groups and fed either standard sow milk replacer (SMR) alone (n = 15), SMR supplemented with sialyllactose 9.5 g/kg (SL, n = 16) or a combination of SL and 6'-sialyllactosamine 9.5 g/kg (SL/SLN, n = 15) for 35 days. Brain spectra were acquired using a 3T Magnetic Resonance Spectroscopic (MRS) system.Results: SMOS fed piglets were observed to have significantly increased the absolute levels of myo-inositol (mIns) and glutamate + glutamine (Glx), in particular, the SL/SLN group. Similar findings were found in the relative amount of these metabolites calculated as ratios to creatine (Cr), choline (Cho) and N-acetylaspartate (NAA) respectively (P < .05). In addition, there were significant positive correlations of brain NAA, total NAA (TNAA), mIns, total Cho (TCho), total Cr (TCr), scyllo-Inositol (SI) and glutathione (Glth) with total white matter volume; Glu and SI with whole brain volume; and SI with whole brain weight respectively (P < .01). SLN and 3'SL intake were closely correlated with the levels of brain Glu, mlns and Glx in the treatment groups only (P < .01-.05).Conclusions: We provide in vivo evidences that milk SMOS can alter many important brain metabolites and neurotransmitters required for optimizing neurodevelopment in piglets, an animal model of human infants.