The effect on bone tissue of β-ecdysterone, a type of ecdysteroid found in many plants, has not been previously investigated. In this study, we found that β-ecdysterone treatment significantly induced alkaline phosphatase (ALP) activity in mesenchymal stem cells in a dose-dependent manner. Real-time polymerase chain reaction (PCR) showed that Runx2, osteocalcin, and type I collagen expression also increased. ICI182780, a specific estrogen receptor antagonist, inhibited the upregulation of ALP activity. Moreover, β-ecdysterone promoted estrogen receptor (ER) reporter gene activity; however, ICI182780 reversed its effect, suggesting that β-ecdysterone has stimulatory effects on osteogenic differentiation via the ER. Furthermore, β-ecdysterone alleviated osteoporosis symptoms in a mouse model without obvious side effects. Therefore β-ecdysterone may be a promising candidate drug for the treatment of osteoporosis.