The seed of Cuscutae Semen has been used as a functional food to prevent osteoporosis and aging, and improve sexual function in Traditional Chinese Medicine. However, there is a little report on its beneficial effects on osteoporosis. The purpose of our study was to explore whether Cuscutae Semen polysaccharide (CSP) could prevent osteoporosis induced by estrogen deficiency in the ovariectomized rat model. The preventive effect of CSP was assessed using the ovariectomized (OVX) rat model by treatment with vehicle or CSP for 12 weeks. Serum indexes related to osteogenesis were measured using ELISA kits. The underlying mechanism of action of CSP was evaluated by qRT-PCR. The findings showed that CSP exerted bone protective effects via the increase of bone mass, BMD, IGF, TGF-β, osteocalcin, and osteoprotegerin, and the decrease of TRAP and CTX levels in estrogen deficiency-induced osteoporosis, which is mediated by up-regulating the expression levels of Osterix, BMP-2, Runx2, and Smad5 and down-regulating the expression levels of TRAP, NFATc1, c-Fos, and cathepsin K. These findings suggested that CSP exhibited the preventive effects in the estrogen deficiency-induced osteoporosis via promoting bone formation and inhibiting bone resorption. Therefore, CSP may be developed as a promising agent for the prevention of estrogen deficiency-induced osteoporosis.