AbstractBone remodeling is vital to the maintenance of bone homeostasis and may lead to destructive skeletal diseases once the balance is disrupted. Crosstalk between Wnt and estrogen receptor (ER) signaling has been proposed in bone remodeling, but the underlying mechanism remains unclear. This study was designed to explore the effect of Wnt‐ER signaling during the osteogenic differentiation of bone marrow stromal cells (BMSCs). Rat BMSCs were isolated and identified using flow cytometry and stimulated with Wnt3a. Wnt3a treatment promoted osteogenic differentiation and mineralization of the BMSCs. Meanwhile, Wnt3a enhanced the expression of ERα as well as the canonical Wnt signaling mediator β‐catenin and the alternative Wnt signaling effector Yes‐associated protein 1 (YAP1). Interestingly, DNA pulldown assay revealed direct binding of transcriptional enhanced associate domain 1 (TEAD1) and lymphoid enhancer binding factor 1 (LEF1), transcriptional partners of YAP1 and β‐catenin, respectively, to the promoter region of ERα. In addition, inhibition of TEAD1 and LEF1 suppressed Wnt3‐promoted BMSC osteogenic differentiation and blocked Wnt3a‐induced ERα expression. Furthermore, an in vivo model of femoral bone defect also supported that Wnt3a facilitated bone healing in an ERα‐dependent way. Together, we suggest that Wnt3a promotes the osteogenic activity of BMSCs through YAP1 and β‐catenin‐dependent activation of ERα, via direct binding of TEAD1 and LEF1 to the ERα promoter.