Background: Periodontitis is a chronic inflammatory disease initiated by bacteria and their virulence factors. Bortezomib (BTZ) is the first proteasome inhibitor for clinical treatment of malignancies. Its anticancer activity is accompanied by an anti-inflammatory effect. However, there are few reports about its anti-inflammatory effect and underlying mechanism in periodontal disease, especially on human periodontal ligament cells (hPDLCs), which are considered a promising cell-based therapy for treating periodontitis. Methods: hPDLCs were treated with lipopolysaccharide (LPS) and pre-treated with BTZ. mRNA and protein levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6, and IL-8 were determined. The anti-inflammatory mechanism of BTZ was studied. Further, experimental rat periodontitis was induced with ligature and LPS injection, and simultaneously and locally treated with BTZ (three injections/week). Four weeks after treatment, microcomputed tomography, immunohistochemical, and histopathologic analyses were performed. Results: Bortezomib administration at safe concentrations (<= 1 nM) inhibited production of proinflammatory cytokines in LPS-stimulated hPDLCs via nuclear factor (NF)-kappa B, p38/extracellular signal-regulated kinase, and mitogen-activated protein kinase/activator protein-1 pathways. Moreover, in the LPS and ligature-induced periodontitis rat model, BTZ suppressed expression of TNF-alpha, IL-1 beta, IL-6, and IL-8, reduced the ratio of receptor activator of NF-kappa B ligand/osteoprotegerin, and prevented alveolar bone absorption. Conclusion: These findings demonstrate the anti-inflammatory activity of BTZ against periodontal inflammatory response and present BTZ as a promising therapy for periodontal disease.