Abstract: Objective To investigate the role of NADPH oxidase (Nox) in the oxidative stress injury of human dermal fibroblasts (HFbs). Methods An oxidative stress injury model was established in HFbs by exposure to H2O2. Normal HFbs and HFbs exposed to H2O2 with and without pretreatment with NADPH oxidase inhibitor were tested for cell viability using MTT assay, and the intracellular reactive oxygen species (ROS) were determined with a DCFH-DA fluorescent probe. Western blotting was used to measure the protein expressions of membrane-bound subunit gp91phox of NADPH oxidase in the cells. Result H2O2 time- and concentration-dependently induced oxidative stress injury in the fibroblasts, causing a reduction of the cell viability to 40% after a 24-h exposure at 700 μmol/L (P<0.05) and an increase of ROS by 2 folds after a 2-h exposure at 700 μmol/L (P<0.05). Compared with the cells with oxidative stress injury, the cells with NADPH oxidase inhibitor pretreatment showed a 20% higher cell viability (P<0.05) and normal ROS level (P<0.05) following H2O2 exposure. Western blotting demonstrated increased expression of gp91phox in the cells exposed to increasing H2O2 concentrations, but gp91phox expression remained normal in cells pretreated with NADPH oxidase inhibitor. Conclusion H2O2 can induce oxidative stress injury in the fibroblasts by affecting NADPH oxidase, especially its membrane-bound subunit gp91phox.