Orthodontically-induced inflammatory root resorption (OIIRR) is one of the procedure-related adverse effects that occur during orthodontic treatment, and the incidence is related to the proinflammatory cytokine produced in response to the mechanical stress caused by the procedure. It is known that tumor necrosis factor (TNF)-α is produced following environmental insults in vivo at an early stage, and that it deeply affects inflammatory bone resorption. However, the relationship between the TNF-α level and root resorption is unclear. In this study, the relationship between TNF-α and root resorption was evaluated using an experimental mouse tooth movement model and a pressure side model using human periodontal ligament (hPDL) cells, as well as an osteoclast culture system. Nine days after the tooth movement in the mouse model, an increase in TNF-α and receptor activator of nuclear factor-κB ligand (RANKL) positive cells was observed. In vitro, their levels increased in the cultures of hPDL cells exposed to the 4 g/cm² pressure. In addition, the differentiation of osteo/odontoclasts was promoted by TNF-α weakly, but the ability to resorb the dentin was unchanged. However, the activation of osteo/odontoclastogenesis is more potent in the presence of RANKL and TNF-α, which leads to synergistic activation. These results suggest that TNF-α may be an aggravating factor for root resorption during orthodontic treatment.