Osteoblasts are bone-forming mesenchymal cells, while macrophages are cells of hematopoietic origin responsible for innate immunity. Lipopolysaccharide (LPS) can induce tolerance in macrophages, whereas interferon (IFN)-gamma can activate macrophages to produce cytokines, exert bactericidal effects, and present antigens. In this study, we examined such macrophagic phenotypes regulated by LPS and IFN-gamma in murine osteoblasts. In both primary calvarial osteoblasts and osteoblastic MC3T3-E1 cells, LPS pretreatment resulted in reduced production of IL-6 in response to a subsequent LPS stimulation or to Salmonella infection, indicating the existence of LPS-induced tolerance in osteoblasts. Furthermore, IFN-gamma treatment of MC3T3-E1 cells resulted in both enhanced IL-6 production in response to LPS and upregulation of major histocompatibility complex class II (MHC II). Following infection, Salmonella-containing vacuoles (SCVs) were formed in MC3T3-E1 cells, and IFN-gamma pretreatment enhanced bactericidal effects on intracellular Salmonella. Taken together, these observations indicate that osteoblasts can exhibit a subset of phenotypes reminiscent of macrophages in the course of bacterial infection.