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<p>Osteoclasts are the key participants in regulation of bone mass. Low-magnitude high-frequency vibration (LMHFV) has been found to be anabolic to bone <i>in vivo</i>. This study aimed to investigate the effect of LMHFV on osteoclast differentiation <i>in vitro</i>. Murine monocyte cell line RAW264.7 cells in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) were treated with or without LMHFV at 45 Hz (0.3 g) for 15 min day^&#8722;1. Tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and actin ring formation were evaluated. Expression of the osteoclast-specific genes, such as cathepsin K, matrix metallopeptidase-9 (MMP-9) and TRAP, were analyzed using real time-PCR. c-Fos, an osteoclast-specific transcription factor, was determined using Western blot. We found that LMHFV significantly decreased the number of RANKL-induced TRAP-positive MNCs (<i>P</i>&#60;0.01), and inhibited the actin ring formation. The mRNA expression of the cathepsin K, MMP-9 and TRAP were down-regulated by LMHFV intervention (all <i>P</i>&#60;0.001). Furthermore, LMHFV also inhibited the expression of c-Fos protein in the RANKL-treated RAW264.7 cells (<i>P</i>&#60;0.05). Our results suggest that LMHFV can inhibit the RANKL-induced osteoclast differentiation of RAW264.7 cells, which give some new insight into the anabolic effects of LMHFV on bone.</p>