BACKGROUND Mechanical stress such as orthodontic forces can produce mechanical damage and inflammatory reaction in the periodontium. Osteopontin (OPN) is a multifunctional cytokine that has been correlated with periodontal disease progression. Because the periodontal ligament (PDL) can be affected by stress and PDL cells are involved in periodontal destruction and remodeling, we aimed to study the influence of mechanical stress on the expression and regulation of OPN in human PDL (HPDL) cells. METHODS The mechanical stress was generated by continuous compressive force, and the expression of OPN was examined by reverse transcription-polymerase chain reaction and Western analysis. The application of inhibitors was used to examine the mechanism involved. RESULTS Both mRNA and protein expression of OPN significantly increased in a force-dependent manner. Increase of receptor activator of nuclear factor-kappa B ligand (RANKL) was also observed. Interestingly, application of indomethacin could abolish the induction of RANKL but not that of OPN, suggesting the cyclooxygenase-independent mechanism for stress-induced OPN expression. In addition, the upregulation of OPN was diminished by Rho kinase inhibitor but not by cytochalasin B. CONCLUSIONS Mechanical stress affects OPN expression in HPDL cells through the Rho kinase pathway. Because OPN participates in bone resorption and remodeling induced by mechanical and biologic signals, these results suggest the significance of stress-induced OPN in HPDL cells in alveolar bone resorption and remodeling.