To shorten the duration of orthodontic treatment it is important not only to reduce risks such as dental caries, periodontal disease, and root resorption, but also to decrease pain and discomfort caused by a fixed appliance. Several studies have investigated the effect of vibration applied to fixed appliances to accelerate tooth movement. Although it was reported that vibration accelerates orthodontic tooth movement by enhancing alveolar bone resorption, the underlying cellular and molecular mechanisms remain unclear. In this study, we investigated the effects of vibration on osteoclastogenesis in vitro and in vivo. Vibration applied to pre-osteoclast cell line RAW264.7 cells enhanced cell proliferation but did not affect their differentiation into osteoclasts. Osteocytes in bone are known to be mechanosensitive and to act as receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL). Therefore, in the present study, vibration was applied to cells from the osteocyte-like cell line MLO-Y4. In MLO-Y4 cells, vibration induced phosphorylation of the inhibitor of NF-κB (IκB) and caused nuclear localization of NF-κB p65. Additionally, vibration increased RANKL mRNA expression, but did not affect osteoprotegerin (OPG) mRNA expression in MLO-Y4 cells, thus resulting in an increased RANKL/OPG ratio. Consistent with these findings, vibration applied during experimental tooth movement increased NF-κB activation and RANKL expression in osteocytes on the compression side of alveolar bone in vivo, whereas vibration had no such effects on the tension side. Furthermore, in a co-culture of MLO-Y4 cells and RAW264.7 cells, vibration applied to MLO-Y4 cells enhanced osteoclastogenesis. These findings suggest that vibration could accelerate orthodontic tooth movement by enhancing osteoclastogenesis through increasing the number of pre-osteoclasts and up-regulating RANKL expression in osteocytes on the compression side of alveolar bone via NF-κB activation.