Objective：To investigate the effect of canonical Wnt signaling antagonist Dickkopf1（Dkk1） on bone morphogenetic protein 9（BMP9） induced C3H10T1/2 mesenchymal stem cells（MSCs） osteogenic differentiation. Methods：C3H10T1/2 cells were infected by recombinant adenovirus expressing green fluorescent protein（GFP），BMP9 and/or Dkk1. Early osteogenic marker，alkaline phos－phatase（ALP） activity was detected by ALP activity assay and staining. Later osteogenic marker calcium deposition was determined by Alizarin Red S staining. Western blot was used to detect the middle osteogenic marker osteopontin（OPN） and osteocalcin（OC） at protein level. Ectopic bone formation assay was carried out to make sure the influence of Dkk1 on BMP9 induced osteogenic differen－tiation of mesenchymal stems cells in vivo. Results：Dkk1 effectively inhibited BMP9 induced ALP activity，calcium deposition，OPN and OC protein expression in C3H10T1/2 cells. Athymic nude mice subcutaneous injection C3H10T1/2 cells showed that Dkk1 inhibited BMP9 induced ectopic bone formation and calcification degree in vivo. Conclusions：Wnt signaling antagonist Dkk1 can dra－matically inhibit BMP9 induced C3H10T1/2 cells osteogenic differentiation and BMP9 induced osteogenic differentiation may require functional canonical Wnt signaling.