Objective To observe the anti-osteoporotic activity of representative phenolic glycosides (curculigoside, curculigine A, orcinol glucoside and orcinol rhamnoside) in Curculigo orchioides on osteoblasts in neonatal rat calvaria and on osteoclasts induced by myelomonocytes. Methods The proliferation of osteoblasts and osteoclasts were determined by MTT assay. The alkaline phosphatase (ALP) activity of osteoblasts and tartrate-resistant acid phosphatase (TRAP) of osteoclasts were detected by using p-PNPP-Na method. The mineralized nodule formation was determined by alizarin red-s (AR-S) staining. TRAP-positive multinucleated cells were counted through TRAP staining. The cytoskeleton of osteoblasts and F-actin ring of osteoclasts were labeled with rhodamine-conjugated phalloidin, and observed under confocal laser scanning microscope. The areas of osteoclastic bone resorption pit in co-culture of osteoblasts and osteoclasts were determined using computer image process. Results Curculigoside (10^-9 mol/L and 10^-8mol/L) significantly promoted the proliferation of osteoblasts and inhibited the TRAP activity of osteoclasts at the concentration of 10^-7-10^-5 mol/L (P<0.05). Curculigoside, orcinol glucoside and orcinol rhamnoside significantly decreased the number of osteoclasts (P<0.05). Curculigoside (10^-10mol/L), curculigine A (10^-9mol/L), orcinol glucoside (10^-9mol/L) and orcinol rhamnoside (10^-9mol/L) significantly increased the ALP activity and bone mineralized nodule formation of osteoblasts (P<0.01), reduced the injury of cytoskeleton of osteoblast induced by 1, 25-(OH)₂-VD₃, decreased the pit area formed by osteoclasts, and destroyed F-actin cytoskeleton structure and pseudopodia of osteoclasts. Conclusion Curculigoside, curculigine A, orcinol glucoside and orcinol rhamnoside, phenolic glycosides extracted from Curculigo orchioides, can promote osteoblastic bone formation and inhibit osteoclastic bone resorption, demonstrating a significant anti-osteoporotic activity.