Glucocorticoids (GCs) are frequently used for the suppression of inflammation in chronic inflammatory diseases. Excessive GCs usage is greatly associated with several side effects, including gingival ulceration, the downward migration of the epithelium, attachment loss and disruption of transeptal fibers. The mechanisms underlying GCs-induced impairments in gingival tissue remains poorly understood. Mitochondrial dysfunction is associated with various oral diseases, such as chronic periodontitis, age-related alveolar bone loss and hydrogen peroxide-induced cell injury in gingival. Here, we reported an unexplored role of cyclophilin D (CypD), the major component of mitochondrial permeability transition pore (mPTP), in dexamethasone (Dex)-induced oxidative stress accumulation and cell dysfunctions in gingival tissue. We demonstrated that the expression level of CypD significantly increased under Dex treatment. Blockade of CypD by pharmaceutical inhibitor cyclosporine A (CsA) significantly protected against Dex-induced oxidative stress accumulation in gingival tissue. And the protective effects of blocking CypD in Dex-induced gingival fibroblasts dysfunction were evidenced by rescued mitochondrial function and suppressed production of reactive oxygen species (ROS). In addition, blockade of CypD by pharmaceutical inhibitor CsA or gene knockdown also restored Dex-induced cell toxicity in HGF-1 cells, as shown by suppressed mitochondrial ROS production, increased CcO activity and decreased apoptosis. We also suggested a role of oxidative stress-mediated p38 signal transduction in this event, and antioxidant N-acety-l-cysteine (NAC) could obviously blunted Dex-induced oxidative stress. These findings provide new insights into the role of CypD-dependent mitochondrial pathway in the Dex-induced gingival injury, indicating that CypD may be potential therapeutic strategy for preventing Dex-induced oxidative stress and cell injury in gingival tissue.