MicroRNAs (miRs) play an essential role in the regulation of bone formation and homeostasis. miR-185 has been reported to negatively regulate osteogenesis in vitro. However, whether it has an impact on in vivo bone homeostasis remains unknown. Here, we demonstrated that primary osteoblasts and mesenchymal stem cells derived from miR-185-knockout (KO) mice exhibited enhanced osteogenesis. Further, we constructed an ovariectomized mouse model to investigate the role of miR-185 during osteoporosis. Micro-computed tomography revealed an increased bone volume in KO compared to wild-type mice 6 weeks after surgery, indicating redundant bone formation after miR-185 depletion. Dual-luciferase reporter assays identified biglycan (Bgn), which promotes bone formation through the BMP/Smad pathway, as the direct target of miR-185. Taken together, these findings indicate that blocking miR-185 expression increases bone formation during osteoporosis, which may partly occur through the regulation of Bgn expression and BMP/Smad signaling.