<jats:sec><jats:title>Background and Objective</jats:title><jats:p>Urokinase‐plasminogen activator (<jats:styled-content style="fixed-case">uPA</jats:styled-content>) is a serine protease expressed at high basal level in normal gingival cervical fluid. Despite its known pathologic role in tissue proteolysis in periodontitis, little is known concerning <jats:styled-content style="fixed-case">uPA</jats:styled-content> physiological function in oral tissue. Recent evidence in cancer cells has implicated the <jats:styled-content style="fixed-case">uPA</jats:styled-content> system in <jats:styled-content style="fixed-case">DNA</jats:styled-content> repair and anti‐apoptotic pathways. This study is aimed to evaluate the protective function of urokinase against oxidative <jats:styled-content style="fixed-case">DNA</jats:styled-content> damage in periodontal ligament (<jats:styled-content style="fixed-case">PDL</jats:styled-content>) fibroblast, and to propose a new biological role for <jats:styled-content style="fixed-case">uPA</jats:styled-content> in oral cavity.</jats:p></jats:sec><jats:sec><jats:title>Material and Methods</jats:title><jats:p>PDL cells were isolated from human wisdom teeth obtained from healthy donors. An oxidative stress model was created in which <jats:styled-content style="fixed-case">PDL</jats:styled-content> cells were incubated with 20, 30, 40 and 60 μmol/L hydrogen peroxide. Twenty‐four hours before and after peroxide treatment, cells were treated with <jats:styled-content style="fixed-case">uPA</jats:styled-content> and amiloride. Cell viability was assessed by <jats:styled-content style="fixed-case">3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5diphenyltetrazolium bromide</jats:styled-content> assay, apoptosis by <jats:styled-content style="fixed-case">DAPI</jats:styled-content>‐staining and annexin V/propidium iodide assay, and <jats:styled-content style="fixed-case">DNA</jats:styled-content> breaks by alkaline comet assay. For estimating <jats:styled-content style="fixed-case">DNA</jats:styled-content> damage level, γ‐H2<jats:styled-content style="fixed-case">AX</jats:styled-content> expression was studied using flow cytometry and immunostaining.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>The incubation of the peroxide‐treated cells with <jats:styled-content style="fixed-case">uPA</jats:styled-content> significantly increased cell viability and decreased apoptosis. A significant decrease in the number of γ‐H2<jats:styled-content style="fixed-case">AX</jats:styled-content> foci was seen at 30 μmol/L hydrogen peroxide in <jats:styled-content style="fixed-case">uPA</jats:styled-content>‐treated cells. <jats:styled-content style="fixed-case">uPA</jats:styled-content> inhibition as a result of amiloride treatment, in turn, induced a reduction in cell viability. In addition, there was a significant decrease in the levels of <jats:styled-content style="fixed-case">DNA</jats:styled-content> damage in <jats:styled-content style="fixed-case">uPA</jats:styled-content>‐treated groups as measured by the comet assay.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>The present study brings support to the theory that uPA may have a protective role for periodontal tissue and could protect PDL fibroblasts from oxidative <jats:styled-content style="fixed-case">DNA</jats:styled-content> damage and apoptosis.</jats:p></jats:sec>