Although mouse liver F4/80⁺ Kupffer cells consist of cytokine-producing CD11b⁺ cells and phagocytic CD68⁺ cells, an undefined CD11b⁻ CD68⁻ subset (30%) also exists. We herein demonstrate a more fundamental classification by adding CD32 (FcγRII), which covers most liver F4/80⁺ cells and the distinct functions of them. Among the F4/80⁺ cells, 50%, 40%, and 30% of cells were CD32⁺, CD68⁺, and CD11b⁺, respectively, and one-half of the CD68⁺ cells coexpressed CD32. CD68⁺ and CD32⁺ cells, but not CD11b⁺ cells, expressed a phagocytosis-related CRIg. Gy (6) irradiation depleted liver CD11b⁺ cells and those in the spleen, bone marrow, and peripheral blood but not liver CD32/CD68⁺ cells. Transfer of bone marrow cells into the irradiated mice reconstituted liver CD11b⁺ cells. Conversely, clodronate pretreatment depleted only liver CD32/CD68⁺ cells but not liver CD11b⁺ cells and peripheral blood or spleen CD11b⁺ monocytes/macrophages. Moreover, the CD32⁺ cells might be precursors of CD68⁺ cells, as a large proportion of CD32⁺ cells expressed the c-kit (CD117), and CD34 and CD32⁺ cells acquired CD68 immediately after bacteria administration. CD32/CD68⁺ cells, but not CD11b⁺ cells, expressed resident macrophage-specific MerTK and CD64 (FcγRI). Challenge with Staphylococcus aureus or liver metastatic EL-4 tumor cells indicated that the CD68⁺ subset is engaged in systemic bactericidal activity, whereas the CD11b⁺ subset is pivotal for liver antitumor immunity. Human liver CD14⁺ Kupffer cells could also be classified into three similar subsets. These results suggest that liver CD68⁺ Kupffer cells and CD11b⁺ Kupffer cells/macrophages are developmentally and functionally distinct subsets.