The subfornical organ is an important regulator of water-salt metabolism and energy balance of the body, involved in the control of the cardiovascular system and immune regulation. The organ comprises several cell populations, among which microglia and macrophages remain uncharacterized. This study aimed at structural, cytochemical, and functional characterization of microglia and macrophages of the subfornical organ in rats. Brain specimens were collected from mature male Wistar rats (n = 8). Microglia and macrophages were revealed by immunostaining with poly- and monoclonal antibodies against calcium-binding protein Iba1 and lysosomal protein CD68; the slides were examined by light and confocal laser microscopy. The study provides a comprehensive morphological characterization of microglial cells and macrophages of the subfornical organ. We demonstrate that the majority of Iba1-expressing cells in this area of the brain are microglial cells, not macrophages. Pre-activated state of the subfornical organ microglia may reflect structural and functional features of this organ and specific functions of local microglia. Subependymal microglial cells, the processes of which penetrate into the third ventricle of the brain, constitute a distinct subpopulation among the Iba1-expressing cells of the subfornical organ. Apart from microglial elements, the subfornical organ contains few tissue macrophages with characteristic strong expression of CD68 accompanied by undetectable or weak expression of Iba1.