Scope: Various biological effects have been postulated for human milk oligosaccharides (HMO), as deduced from in vitro, animal, and epidemiological studies. Little is known about their metabolic fate in vivo in the breast-fed infant, which is presented here. Methods and results: Human milk and infant urine and feces were collected from ten mother child pairs and analyzed by MALDI-TOF MS (/MS), accompanied by high-performance anion-exchange chromatography with pulsed amperometric detection. Previously, we detected intact small and complex HMO in infant urine, which had been absorbed from gut, as verified via intrinsic C-13-labeling. Our current work reveals the presence of novel HMO metabolites in urine and feces of breast-fed infants. The novel metabolites were identified as acetylated HMOs and other HMO-like structures, produced by the infants or by their gut microbiota. The finding of secretor-or Lewis-specific HMO in the feces/urine of infants fed with nonsecretor or Lewis-negative milk suggested a correspondent modification in the infant. Conclusion: Our study reveals new insights into the metabolism of neutral HMO in exclusively breast-fed infants and provides further indications for multiple factors influencing HMO metabolism and functions that should be considered in future in vivo investigations.