Osteoclasts, bone-resorbing multinucleated cells, are differentiated from hemopoietic progenitors of the monocyte/macrophage lineage. Bone resorption by osteoclasts is considered a potential therapeutic target to the treatment of erosive bone diseases, including osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we found that alisol-B, a phyto-steroid from Alisma orientale Juzepczuk, exhibited inhibitory effects on osteoclastogenesis both in vitro and in vivo. Although RT-PCR analysis showed that alisol-B did not affect the 1alpha,25(OH)(2)D(3)-induced expressions of RANKL, OPG and M-CSF mRNAs in osteoblasts, addition of alisol-B to co-cultures of mouse bone marrow cells and primary osteoblasts with 10(-8)M 1alpha,25(OH)(2)D(3) caused significant inhibition of osteoclastogenesis. We further examined the direct effects of alisol-B on osteoclast precursors. Alisol-B strongly inhibited RANKL-induced osteoclast formation when added during the early stage of cultures, suggesting that alisol-B acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, alisol-B inhibited the phosphorylation of JNK, which are upregulated in response to RANKL in bone marrow macrophages, alisol-B also inhibited RANKL-induced expression of NFATc1 and c-Fos, which are key transcription factors for osteoclastogenesis. In addition, alisol-B suppressed the pit-forming activity and disrupted the actin ring formation of mature osteoclasts. In a hypercalcemic mouse model induced by 2-methylene-19-nor-(20S)-1alpha,25(OH)(2)D(3) (2MD), an analog of 1alpha,25(OH)(2)D(3), administration of alisol-B significantly suppressed 2MD-induced hypercalcemia as resulting from the inhibition of osteoclastogenesis. Taken together, these findings suggest that alisol-B may be a potential novel therapeutic molecule for bone disorders by targeting the differentiation of osteoclasts as well as their functions.