The current study was to evaluate the protective effects of Chinese medicine prescription Zuogui Pill (ZGP) on osteoporosis (OP) in zebrafish larvae induced by dexamethasone. Alizarin Red staining, calcium and phosphorus determination were performed to evaluate the effect of ZGP on bone mineralization. Hydroxyproline (HP), Alkaline phosphatase (ALP), and tartrate resistant acid phosphatase (TRAP) were also measured by commercial kits. We found that ZGP had positive effects in increasing bone mineral content (BMC), strengthening bone biomechanical, promoting bone formation, inhibiting bone resorption, and mediating protein levels of TGF-β1/Smads signaling pathway. The findings demonstrated that ZGP treatments inhibited the phosphorylation of TGF-β and p-Smad 3 as well as the expressions of collagen I and collagen II by western blot. Taken together, we demonstrated that ZGP may prevent osteoporosis via reversing the imbalance of bone fomation/bone resorption and activating the TGF-β-Smad signal.