The E3 ubiquitin ligase TRAF6 [tumor necrosis factor (TNF) receptor (TNFR)-associated factor 6] and the associated kinase TAK1 [transforming growth factor-β (TGF-β)-activated kinase 1] are key components of the signaling pathways that activate nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) in response to various stimuli. The cytokine RANKL (receptor activator of NF-κB ligand) is essential for the differentiation of bone marrow cells into bone-resorbing osteoclasts through the activation of NF-κB and MAPK. We found that the scaffold protein RACK1 (receptor for activated C kinase 1) selectively mediated the RANKL-dependent activation of p38 MAPK through the TRAF6-TAK1 axis by interacting with the MAPK kinase MKK6 (MAPK kinase kinase 6), which is upstream of p38 MAPK. RACK1 was necessary for the differentiation of bone marrow cells into osteoclasts through the stimulation of p38 MAPK activation. Osteoclast precursors exposed to RANKL exhibited an interaction among RACK1, RANK, TRAF6, TAK1, and the kinase MKK6, thereby leading to the activation of the MKK6-p38 MAPK pathway. Experiments in which RACK1 or TAK1 was knocked down in osteoclast precursors indicated that RACK1 acted as a bridge, bringing MKK6 to the TRAF6-TAK1 complex. Furthermore, local administration of RACK1-specific small interfering RNA (siRNA) into mice calvariae reduced the RANKL-induced bone loss by reducing the numbers of osteoclasts. These findings suggest that RACK1 specifies the RANKL-stimulated activation of p38 MAPK by facilitating the association of MKK6 with TAK1, and may provide a molecular target for a new therapeutic strategy to treat bone diseases.