Background: We previously found the conditions of supplementary vibration that accelerated tooth movement and induced bone resorption in an experimental rat tooth movement model. However, the molecular biological mechanisms underlying supplementary vibration-induced orthodontic tooth movement are not fully understood. Transforming growth factor (TGF)-β upregulates osteoclastogenesis via induction of the receptor activator of nuclear factor kappa B ligand expression, thus TGF-β is considered an essential cytokine to induce bone resorption. Objectives: The aim of this study is to examine the role of TGF-β during the acceleration of orthodontic tooth movement by supplementary vibration. Materials and methods: In experimental tooth movement, 15 g of orthodontic force was loaded onto the maxillary right first molar for 28 days. Supplementary vibration (3 g, 70 Hz) was applied to the maxillary first molar for 3 min on days 0, 7, 14, and 21. TGF-β receptor inhibitor SB431542 was injected into the submucosal palatal and buccal areas of the maxillary first molars once every other day. The co-culture of RAW264.7 cells and MLO-Y4 cells was used as an in vitro osteoclastogenesis model. Results: SB431542 suppressed the acceleration of tooth movement and the increase in the number of osteoclasts by supplementary vibration in our experimental rat tooth movement model. Immunohistochemical analysis showed supplementary vibration increased the number of TGF-β1-positive osteocytes in the alveolar bone on the compression side during the experimental tooth movement. Moreover, vibration-upregulated TGF-β1 in MLO-Y4 cells induced osteoclastogenesis. Conclusions: Orthodontic tooth movement was accelerated by supplementary vibration through the promotion of the production of TGF-β1 in osteocytes and subsequent osteoclastogenesis.