Background <p>Breast milk not only is the most optimal source of nutrition for newborns but also provides crucial immune support. While there is extensive evidence highlighting the health benefits of breastfeeding, the scientific understanding behind many of these advantages remains limited. The immune cell fraction of breast milk has been poorly studied, and characterization of this essential component may be crucial to shed light on the subject. Here, we investigate the population of neutrophils in breast milk to unveil their phenotype and functions.</p> Methods <p>Neutrophils in human breast milk were characterized by flow cytometry, and their phenotype was contrasted with that of their circulating counterparts using a paired approach. Transmission electron microscopy (TEM), intracellular granule immunocytochemistry, and Giemsa staining were employed to reveal their intracellular structure. In vitro neutrophil incubations were performed to assess the phenotypic modifications of circulating neutrophils in a breast milk environment. Functional assays were carried out to assess the activity of neutrophils and their potential impact on adaptive immunity. Finally, a proteomic approach was used on isolated neutrophils and their supernatant after cultivation to define the protein-based mechanisms involved in their biology. Paired <i>t</i>-tests and one- and two-way analysis of variance (ANOVA) were used for statistical analyses.</p> Results <p>In the breast milk from the first 5 days (known as colostrum), neutrophils represent most of the cellular compartment. Colostrum neutrophils present a cell surface signature markedly different from circulating neutrophils with a clear CXCR4⁺CD62L⁻CXCR2^mid phenotype and important differences in other markers. These differences are partially phenocopied upon incubation of blood neutrophils with colostrum whey, implying that cell transition occurs owing to the niche environment. Additionally, colostrum neutrophils exhibit reduced cytoplasmic granule content and altered canonical functions. Proteomic analyses reveal that the most important processes in colostrum neutrophils are related to antigen binding and humoral response pathways.</p> Conclusions <p>This study provides evidence of a unique population of neutrophils in early breast milk connected to adaptive immunity, which may hold the key to understanding the mechanisms behind the immunological benefits of breastfeeding.</p>