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Background: Postpartum depression (PPD) is a prevalent psychiatric disorder during the postnatal period, and it exerts adverse impacts not only on mothers but also on infants, impairing the well‐being of the whole family. However, the role of peptides in the breast milk of mothers with PPD has not been studied. The purpose of this study was to unveil the peptidomic profile of PPD from breast milk samples. Methods: We performed comparative peptidomic profiling of human breast milk from PPD and control mothers using liquid chromatography–tandem mass spectrometry technology with iTRAQ‐8 labelling. GO and KEGG pathway analyses of precursor proteins were used to predict the underlying biological functions of differentially expressed peptides (DEPs). Ingenuity Pathway Analysis (IPA) was further performed to explore the interactions and involved pathways of DEPs. Results: A total of 294 peptides from 62 precursor proteins were identified to be differentially expressed in the breast milk of PPD mothers compared with the control group. Bioinformatics analysis predicted that these DEPs were associated with ECM–receptor interaction, neuroactive ligand‐receptor interaction, cell adhesion molecule binding and oxidative stress in macrophages. These results indicate that DEPs from human breast milk may play a part in PPD and become promising noninvasive biomarkers. Peptides from human breast milk were expected to be noninvasive biomarkers for postpartum depression. Bioinformatics analysis of differentially expressed peptides found that the pathogenesis of postpartum depression was closely related to ECM remodelling and macrophage‐mediated inflammation. [ABSTRACT FROM AUTHOR]