The mammary gland is unique in female mammals. Mammary tissue undergoes development and remodeling during lactation, a stage associated with high susceptibility to bacterial infections, inducing an inflammatory condition called mastitis. Although the immune response of the mammary gland has been the subject of intense research to improve prevention and treatment efficacy, the precise definition of its immune composition at this particular physiological stage is still missing. We combined single-cell RNA-Seq, flow cytometry, and three-dimensional confocal microscopy techniques to characterize the immune landscape of lactating murine mammary tissue. Macrophages dominated the immune cell repertoire and could be subdivided into at least two subsets: ductal and stromal macrophages. Ductal macrophages represented approximately 80% of the total CD45 pos immune cells and co-expressed F4/80 and CD11c, with high levels of MHC class II molecules. They were strategically poised below the alveolar basal cells in contact with the myoepithelial cell network. Adaptive T and B lymphocytes were remarkably less numerous at this stage, which could explain the limited efficacy of vaccination against mastitis. These results support the view that new strategies to increase mammary immunity and prevent mastitis should be devised.