The canonical Wnt/beta-catenin signaling pathway in osteoblast-lineage cells inhibits osteoclastogenesis through the expression of osteoprotegerin (Opg), a decoy receptor of receptor activator of Nf-kappa b, (Rank) ligands. Wnt5a, a typical non-canonical Wnt ligand, enhances the expression of Rank in osteoclast precursors, which, in turn, promotes the Rank ligand (Rankl)-induced formation of osteoclasts. In contrast, Wnt16 and Wnt4 have been shown to inhibit the Rankl-induced formation of osteoclasts through non-canonical Wnt signals. However, the relationships among these Wnt ligands in osteoclastogenesis remained to be elucidated. We herein showed that Wnt16, but not Wnt4, inhibited the Rankl-induced osteoclastogenesis in bone marrow-derived macrophage (BMM) cultures. Wnt3a and Wnt4 inhibited the 1 alpha,25-dihydroxy vitamin D-3 (1,25D(3))-induced osteoclastogenesis in co-cultures prepared from wild-type mice, but not in those from Opg(-/-) nice. Wnt16 inhibited the 1,25D(3)-induced formation of osteoclasts in both wild-type and Opg(-/-) co-cultures. Wnt16, Wnt4, and Wnt3a failed to inhibit the pit-forming activity of osteoclasts. Wnt16 failed to inhibit the Wnt5a-induced expression of Rank in osteoclast precursors. In contrast, Wnt5a abrogated the inhibitory effects of Wnt16 on Rankl-induced osteoclastogenesis. These results suggested that Wnt16 inhibited osteoclastogenesis, but not the function of osteoclasts and that Wnt16, an inhibitory Wnt ligand for osteoclastogenesis, regulates bone resorption in conjunction with Wnt5a. (C) 2015 Elsevier Inc. All rights reserved.