The aim of the present study was to compare the osteoclast-inhibiting ability of recombinant osteoprotegerin (OPG) protein (rhOPG-Fc) and recombinant receptor activator of nuclear factor κB (rhRANK) in vitro and in vivo. Osteoclasts were cultured with either rhOPG-Fc or rhRANK for 9 days. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and resorption pits in bone slices were then counted. In the in vivo investigation, female mice were bilaterally ovariectomized (OVX) and intraperitoneally injected with 3 mg/kg rhOPG-Fc or rhRANK for 12 weeks, respectively. Bone metabolism, bone mineral density and microstructure changes were then evaluated. The number of TRAP-positive cells and bone resorption pits decreased significantly following culture with either rhOPG-Fc or rhRANK, and this was more marked following culture with rhRANK compared with rhOPG-Fc. The levels of calcium and alkaline phosphatase in the serum were similar pre-OVX and after 12 weeks of treatment, while the levels of phosphorus in the serum were higher following treatment with rhRANK compared with rhOPG. The bone mineral density (BMD) of the whole body, femoral neck and L4 lumbar vertebral body in the mice treated with either rhOPG-Fc or rhRANK increased markedly. In addition, the mice treated with rhRANK exhibited significantly higher BMD in the femoral neck and lumbar vertebral body compared with those treated with rhOPG-Fc. Microcomputed tomography analysis demonstrated that the mice treated with rhRANK exhibited an increased bone volume and structure model index, and decreased trabecular spacing compared with those treated with rhOPG-Fc. rhRANK increased the inhibition of osteoclast differentiation and bone resorption, and rescued OVX-induced osteoporosis more effectively compared with rhOPG-Fc.