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The objective of this study was to explore the effect of SMO16, a Korean poly-herbal medicine, on bone mass in ovariectomized (OVX) rats. The loss of bone mass is known to be associated with hormone deficiency in OVX rats. In the present study, we successfully produced the osteoporosis animal-model by dissecting the ovary. To determine the protective effect of SMO16 in OVX rat, SMO16 was administered at a dose of 2.25 g/kg in saline solution for 8 weeks after OVX. Furthermore, we evaluated the direct effect of SMO16 by using a soft X-ray system and Masson’s trichrome staining, phloxine-tartrazine staining, and immunohistochemistry in vivo. SMO16 significantly regulated the expression of proteoglycan and osteopontin. These data suggest that SMO16 exerts a protective effect against osteoporosis via the activation of proteoglycan and osteopontin that are closely associated with bone balance. Moreover, these findings suggest that SMO16 can act as a potential therapeutic agent for the treatment of osteoporosis.