Updated project metadata. Macrophages are involved in immune defense, organogenesis and tissue homeostasis. They also contribute to the different phases of mammary gland remodeling during development, pregnancy and involution post-lactation. Yet, less is known about the dynamics of mammary gland macrophages in the lactation stage. Here, we describe a macrophage population present during lactation in mice. By multi-parameter flow cytometry and single-cell RNA sequencing we reveal this population as distinct from the two resident macrophage subsets present pregestationally. These lactation-induced macrophages (LiMacs) are predominantly monocyte-derived and expand by proliferation in situ concomitant with nursing. LiMacs develop independently of IL-34 but require CSF-1 signaling and are partly microbiota-dependent. Locally, they reside adjacent to the basal cells of the alveoli and extravasate into the milk. Moreover, we also found several macrophage subsets in human milk, resembling LiMacs. Collectively, these findings reveal the emergence of unique macrophages in the mammary gland and milk during lactation.