Abstract: Objective To understand the mechanism by which tenascin-C regulates osteoblast differentiation and the role of tenascin-C in osteoporosis. Methods Tenascin-C protein expression in femoral spongy bone of mice with or without osteoporosis was analyzed using Western blotting. In MC3T3-E1 osteoblasts with or without tenascin-C depletion by a specific siRNA targeting tenascin-C, alkaline phosphatase activity and Dickkopf-1 (DKK-1) expression were determined using quantitative RT-PCR and Western blotting, and the transcriptional activity of Wnt signaling pathway was analyzed using a luciferase reporter assay. The possible interaction of tenascin-C with DKK-1 predicted by STRING software was verified by immunoprecipitation. Results Tenascin-C was markedly down-regulated in hemoral spongy bone of mice with osteoporosis as compared with the control mice. Osteoblastic differentiation was markedly suppressed in MC3T3-E1 osteoblast after tenascin-C depletion, and was significantly reversed by simultaneous β-catenin over-expression. siRNA-mediated knockdown of tenascin-C, which bound DKK-1, up-regulated the expression of DKK-1 and consequently lowered the transcriptional activity of Wnt pathway. Conclusion Tenascin-C knockdown attenuates its negative control on DKK-1 to suppress the transcriptional activity of Wnt pathway, which in turn suppresses osteoblastic differentiation and promotes osteoporosis.