The activities of osteoclasts and osteoblasts are balanced to maintain normal bone density. Many pathological conditions cause osteoclastic bone resorption in excess of osteoblastic bone formation, resulting in osteoporosis. We found that oral administration of Artemisia annua ethanol extract (AaE) or major components, artemisinin and arteannuin B, to ovariectomized (OVX) mice prevented bone loss, as verified by examining three-dimensional images and bone morphometric parameters derived from microcomputed tomography analysis, as well as serum levels of bone turnover markers and proinflammatory cytokines. The administered doses were not toxic to the liver or kidney and showed promising effects that were comparable to those of 17β-estradiol treatment. At non-cytotoxic concentrations, AaE and active components, artemisinin, artemisinic acid, and arteannuin B, potently inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis and the formation of osteoclast-mediated resorption pits. Furthermore, AaE, artemisinin, and arteannuin B remarkably reduced the expression of the c-Fos and NFATc1 transcription factors, which play critical roles in RANKL-induced osteoclast differentiation. Taken together, the in vivo anti-osteoporotic activity of AaE may be derived from the anti-osteoclastic and anti-bone resorptive activities of its active components. AaE has beneficial applications for the prevention and inhibition of osteoporosis and osteoclast-mediated bone diseases.