ABSTRACT Breast milk proteome comprises hundreds of bioactive proteins supporting infant development. The extent to which maternal metabolic conditions modify the proteome is poorly known. This study investigates proteome evolution from colostrum to mature milk and examines the impacts of maternal gestational diabetes mellitus (GDM) and BMI on the proteome. We analyzed the proteome by data‐independent acquisition (DIA)‐based LC‐MS/MS from colostrum and mature milk samples collected from mothers ( n = 47) with varying BMI values and with ( n = 11) or without ( n = 36) GDM. We identified 3496 proteins, of which 1055 were differentially abundant between colostrum and mature milk. Colostrum was enriched in proteins related to MHC II antigen response, intestinal IgA, adhesion, and glycosylation, while mature milk showed enrichment in lipid biosynthesis, protein translation, and degradation. In mature milk, mothers with both GDM and higher BMI had increased levels of proteins related to HDL formation. Proteins supporting the maturation of the naive gut immune system were prevalent in colostrum, while those prevalent in mature milk reflected the mammary gland's effort in biosynthesis. Both BMI and GDM have measurable effects on the breast milk proteome. The clinical significance of these findings on a child's future health needs to be clarified.