Postmenopausal osteoporosis is a common bone metabolic disease, and gut microbiota (GM) imbalance plays an important role in the development of metabolic bone disease. Here, we show that ovariectomized mice had high levels of lipopolysaccharide in serum and gut microbiota dysbiosis through increases in luminal Firmicutes:Bacteroidetes ratio. We depleted the GM through antibiotic treatment and observed improvements in bone mass, bone microstructure, and bone strength in ovariectomized mice. Conversely, transplantation of GM adapted to ovariectomy induced bone loss. However, GM depletion reversed ovariectomy-induced gene expression in the tibia and increased periosteal bone formation. Furthermore, bioinformatics analysis revealed that the G-protein-coupled bile acid receptor (TGR5) and systemic inflammatory factors play key roles in bone metabolism. Silencing TGR5 expression through small interfering RNA (siRNA) in the local tibia and knockout of TGR5 attenuated the effects of GM depletion in ovariectomized mice, confirming these findings. Thus, this study highlights the critical role of the GM in inducing bone loss in ovariectomized mice and suggests that targeting TGR5 within the GM may have therapeutic potential for postmenopausal osteoporosis.