Staphylococcus aureus osteomyelitis leads to extensive bone destruction. Osteoclasts are bone resorbing cells that are often increased in bone infected with S. aureus. The cytokine RANKL is essential for osteoclast formation under physiological conditions but in vitro evidence suggests that inflammatory cytokines may by-pass the requirement for RANKL. The goal of this study was to determine whether RANKL-dependent osteoclast formation is essential for the bone loss that occurs in a murine model of S. aureus osteomyelitis. To this end, humanized-RANKL mice were infected by direct inoculation of S. aureus into a unicortical defect in the femur. Mice were treated with vehicle or denosumab, a human monoclonal antibody that inhibits RANKL, both before and during a 14-day infection period. The severe cortical bone destruction caused by infection was completely prevented by denosumab administration even though the bacterial burden in the femur was not affected. Osteoclasts were abundant near the inoculation site in vehicle-treated mice but absent in denosumab-treated mice. In situ hybridization demonstrated that S. aureus infection potently stimulated RANKL expression in bone marrow stromal cells. The extensive reactive bone formation that occurs in this osteomyelitis model was also reduced by denosumab administration. Lastly, there was a notable lack of osteoblasts near the infection site suggesting that the normal coupling of bone formation to bone resorption was disrupted by S. aureus infection. These results demonstrate that RANKL-mediated osteoclast formation is required for the bone loss that occurs in S. aureus infection and suggest that disruption of the coupling of bone formation to bone resorption may also contribute to bone loss in this condition.