Human milk is often the sole dietary source for the first few months in life. It contains all the nutrients necessary for the infant to thrive, but also ingredients that may provide health benefits beyond those of traditional nutrients. Human milk oligosaccharides (HMO) comprise part of these functional ingredients; 1 L of mature human milk contains approximately 5-10 g unbound oligosaccharides, and >130 different HMO have been identified. Both their high amount and structural diversity are unique to humans. Only trace amounts of these oligosaccharides are present in mature bovine milk and, as a consequence, in bovine milk-based infant formula. The potential health benefits of HMO that were uncovered over the years may affect breast-fed infants both locally and systemically. Recent advances in glycobiology and nutrition, including the use of stable isotopes, frontal-affinity chromatography, glycan microarrays, MS, and automated solid-phase carbohydrate synthesis, will help verify hypotheses and unravel the mysteries behind HMO.