BackgroundHuman milk oligosaccharides (HMOs) and other milk-derived metabolites are crucial for infant health, influencing gut microbiota and overall development. ObjectiveThis study aims to uncover insights into the variations of HMOs and non-HMO metabolites based on secretor (Se) status, lactation time, mode of delivery, and infant sex. MethodsAn exploratory cross-sectional study was designed to compare the levels of HMOs and non-HMOs metabolites in milk samples from 129 lactating Chinese women within 1 year postpartum. Nuclear magnetic resonance analysis was employed for the identification and quantification of the metabolites. The metabolites measured were grouped into sugars, free amino acids, fatty acids, and metabolites related to energy metabolism. The influence of delivery mode and infant sex on milk metabolite composition were explored. ResultsUniform Manifold Approximation and Projection (UMAP) analysis of HMOs profiles revealed distinct clustering based on Se status, with significant differences in 2'-FL and 3-FL levels observed between Se+ and Se- groups. A decreasing trend for 2'-FL and 6-’SL levels, along with an increase in 3-FL levels, was observed with increasing lactating period within 12 months postpartum. Non-HMOs metabolite analysis indicated that Se status only affected glutamate levels. An increase in glutamine levels was observed 3–9 months postpartum. A continuous increase in o-phosphocholine levels was noted in 12 months postpartum, along with reductions in citrate and sn-glycero-phosphocholine levels. Delivery mode and infant sex did not affect both HMOs and non-HMOs levels. ConclusionsMetabolomic analysis of human milk reveals significant variation of HMOs, but not in non-HMOs, based on Se status. Changes in certain HMOs and non-HMOs levels were also observed over the one year of lactation. Understanding how these metabolites change over time may influence recommendations for maternal diet, supplementation, and the timing of breastfeeding to ensure optimal nutrient delivery to the infant.