To clarify the role of calpain in the receptor activator of NF-B ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on ␣-spectrinolysis and that -calpain, rather than m-calpain, was activated during RANKLsupported osteoclastogenesis in RAW 264.7 cells. Overexpression of -calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-B activation via inhibition of the cleavage of inhibitor of NF-B (IB␣) in RAW 264.7 cells. Taken together, our findings suggest that -calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-B activation.