PURPOSE: This study aimed to evaluate whether maternal nutritional status, maternal age, mode of delivery, and the infant's sex influence the profiles of amino acids, energetic metabolites, sugars, and fatty acids and as well as the metabolic pathways in mature human milk human milk (HM). METHODS: This was a cross-sectional, prospective, and observational study. HM samples from normal weight (NW, n = 60), overweight (OW, n = 35), and obese (OB, n = 14) women were analyzed using a non-targeted GC-MS method to identify the metabolome. Data obtained were analyzed with Metaboanalyst software (v. 5.0) and SPSS (v.25.0). RESULTS: OB women HM contains a higher proportion of amino acids such as leucine, lysine, tyrosine, and aspartic acid, energy metabolites such as lactic and succinic acid, and sugars and derivatives such as fucose, rhamnose, and gluconic acid (p <
0.05) compared with normal weight women HM. HM from women >
25 years of age contains a lower proportion of lauric acid and a higher proportion of leucine and tyrosine (p <
0.05) than ≤ 25 years women HM. Also, HM intended for female infants has a higher leucine and gluconic acid content. The main altered metabolic pathways in OB women HM correspond to amino acids and energetic metabolism. CONCLUSION: OB women HM provides more amino acids, energetic molecules, and sugars. Increased maternal weight, BMI, and body fat mass predispose to more leucine and aspartic acid in HM. Maternal age influences lauric acid, leucin, and tyrosine levels, while the infant's sex influences leucine and gluconic acid levels in HM. The impact of obese women's HM metabolome on the offspring's physiology needs to be explored. TRIAL REGISTRATION: R-2021-785-096.