We have investigated the effects of technetium‐99 conjugated with methylene diphosphonate (99Tc‐MDP), an agent for radionuclide therapy, on receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis, and explored its underlying mechanisms. A murine macrophage cell line RAW264.7 and bone marrow derived‐macrophages from C57BL/6 mice (BMMs) were used as models for osteoclastogenesis in vitro. The expression of some key factors in RANKL (50 ng/ml)‐induced osteoclastogenesis from RAW264.7 was investigated by flow cytometry and real‐time RT‐PCR. To detect multinucleated osteoclast formation, RAW264.7 was induced with RANKL for four days and BMMs were induced by RANKL (50 ng/ml) and macrophage colony‐stimulating factor (M‐CSF, 20 ng/ml) for seven days, and the cells were then stained with tartrate‐resistant acid phosphatase (TRAP). Osteoclast markers including CD51, matrix metalloproteinases 9 (MMP9) and Cathepsin K were used to evaluate osteoclastogenesis. 99Tc‐MDP (0.01 μg/ml) significantly inhibited RANKL‐induced osteoclastogenesis without any cytotoxicity. 99Tc‐MDP also dramatically abolished the appearance of multinucleated osteoclasts. Expressions of transcription factors, c‐Fos and nuclear factor of activated T‐cells (NFATc1), as well as inflammatory factors induced by RANKL were measured by real‐time RT‐PCR. 99Tc‐MDP inhibited the expressions of c‐Fos, NFATc1 and inflammatory factors such as interleukin 6 (IL‐6), tumor necrosis factor α (TNF‐α) and interleukin 1β (IL‐1β). Moreover, 99Tc‐MDP also inhibited the activation of mitogen‐activated protein kinases (MAPKs) in RAW264.7 cells under RANKL stimulation. In conclusion, 99Tc‐MDP possesses anti‐osteoclastogenic activity on RANKL‐induced osteoclast formation. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Blackwell Publishing Asia Pty Ltd