Macrophages are the principal leukocytes of the epididymis and testis, but their origins, heterogeneity, development, maintenance and function are not well understood. In a multi-pronged approach, we found that CD64hiMHCIIlo and CD64loMHCIIhi macrophage populations of epididymis and testis arise sequentially from yolk sac erythro-myeloid progenitors, embryonic hematopoiesis, and nascent neonatal monocytes. While monocytes were the major developmental source of epididymal and testicular macrophages, both populations self-maintain independent of bone marrow hematopoietic precursors. However, during infection, bone marrow-derived circulating monocytes are recruited to both organs, giving rise to inflammatory macrophages that promote tissue damage. Associated damage of the testis of affected men and in rodent models include leukocytic infiltration, edema formation, fibrosis, germ cell loss and reduced androgen levels. The mouse epididymis comprises four distinct regions: the initial segment (IS) which receives the spermatozoa from the testis, caput, corpus and cauda, where sperm are stored and pass to the vas deferens. The epididymis faces opposing immunological challenges. In the IS and caput, local tolerance is required to avoid autoimmune reactions against immunogenic spermatozoa. Conversely, the cauda is a port of entry for bacteria ascending from the urethra. In both mice and human, the magnitude of immune response in the cauda following bacterial infection is much higher than in the proximal parts of the epididymis, often leading to male infertility due to fibrosis and duct obstruction. We show that immune cell populations are strategically positioned along the epididymis and represent a possible key factor to maintain the immunological equilibrium across the organ.