Excessive osteoclast activity can lead to an imbalance between the synthesis and breakdown of bone, with pathologic consequences that include osteoporosis and periodontitis. Thus, controlling osteoclast differentiation and function has significant therapeutic implications. In this study, we investigated the effects of dehydrocostus lactone (DL) on osteoclast differentiation and activation and elucidated the possible mechanisms underlying these processes. DL suppressed osteoclast differentiation by reducing the expression of the nuclear factor of activated T-cells, cytoplasmic 1. When used to challenge differentiated osteoclasts, DL also effectively inhibited their enlargement and resorption activity, and biochemical approaches revealed that DL attenuates osteoclast activation by inhibiting the migration and lysosome biogenesis and secretion via the down-regulation of integrin β3, PKC-β, and autophagy related 5 expression. Furthermore, DL prevented bone destruction in inflammation- and ovariectomy-induced osteolytic mouse models. These results indicate that DL has therapeutic potential to treat bone diseases caused by excessive or hyperactive osteoclasts.—Lee, H. I., Lee, J., Hwang, D., Lee, G.-R., Kim, N., Kwon, M., Lee, H., Piao, D., Kim, H. J., Kim, N. Y., Kim, H. S., Seo, E. K., Kang, D., Jeong, W. Dehydrocostus lactone suppresses osteoclast differentiation by regulating NFATc1 and inhibits osteoclast activation through modulating migration and lysosome function. FASEB J. 33, 9685–9694 (2019). www.fasebj.org.