The scaffolding protein receptor for activated C-kinase 1 (RACK1) mediates receptor activator of nuclear factor κΒ ligand (RANKL)-dependent activation of p38 MAPK in osteoclast precursors; however, the role of RACK1 in mature osteoclasts is unclear. The aim of our study was to identify the interaction between RACK1 and c-Src that is critical for osteoclast function. A RACK1 mutant protein (mutations of tyrosine 228 and 246 residues to phenylalanine; RACK1 Y228F/Y246F) did not interact with c-Src. The mutant retained its ability to differentiate into osteoclasts; however, the integrity of the RANKL-mediated cytoskeleton, bone resorption activity, and phosphorylation of c-Src was significantly decreased. Importantly, lysine 152 (K152) within the Src homology 2 (SH2) domain of c-Src is involved in RACK1 binding. The c-Src K152R mutant (mutation of lysine 152 into arginine) impaired the resorption of bone by osteoclasts. These findings not only clarify the role of the RACK1-c-Src axis as a key regulator of osteoclast function but will also help to develop new antiresorption therapies to prevent bone loss-related diseases.