We have previously shown that mice lacking the TSH receptor (TSHR) exhibit osteoporosis due to enhanced osteoclast formation. The fact that this enhancement is not observed in double null mice of TSHR and TNF suggests that TNF overexpression in osteoclast progenitors (macrophages) may be involved. It is unknown how TNF expression is regulated in osteoclastogenesis. Here, we describe a receptor activator for NFB ligand, RANKL-responsive sequence (RRS: CCG AGA CAG AGG TGT AGG GCC)–spanning from -157 bp to -137 bp of the 5'-flanking region of the TNF gene–that functions as a cis-acting regulatory element. We further show how RANKL treatment stimulates the high mobility group box proteins HMGB1 and HMGB2 to bind the RRS and up-regulates TNF transcription. Exogenous HMGBs elicit the expression of cytokines, including TNF, as well as osteoclast formation. Conversely, TSH inhibits the expression of HMGBs and TNF and the formation of osteoclasts. These results suggest that HMGBs play a pivotal role in osteoclastogenesis. We also show a direct correlation between the expression of HMGBs and TNF and osteoclast formation in TSHR-null mice and TNF-null mice. Taken together, we conclude that HMGBs and TNF play critical roles in the regulation of osteoclastogenesis and the remodeling of bone.