Amelogenin is a novel enamel matrix protein. Knockout mice showed enhanced osteoclast formation and resorption of tooth cementum. This study investigated the effects of amelogenin on osteoclastogenesis. In co-cultures with calvaria osteoblasts and purified bone marrow cells, amelogenin inhibited osteoclastogenesis dramatically. Furthermore, amelogenin inhibited the expression of receptor activator of nuclear factor κB ligand (RANKL), macrophage-colony stimulating factor (M-CSF) and fibronectin in osteoblasts, while RANKL expression was induced by fibronectin and inhibited by treatment with fibronectin small interfering RNA. These results suggest that the inhibitory effects of amelogenin on osteoclastogenesis lead to downregulation of RANKL, M-CSF and fibronectin production in osteoblasts.