Objective To explore the anti-oxidation stress effect of hydrogen-rich water (HW) on thyroid associated ophthalmopathy (TAO). Methods The orbital fibroblasts were derived from orbital adipose connective tissue of TAO patients and cultured in vitro. The cells were treated with different concentrations of hydrogen peroxide (H₂O₂) for 18 h, and the proliferation ability was detected by CCK-8 method to determine the appropriate H₂O₂ concentration. The cells were divided into four groups:blank group (normal culture), H₂O₂ group (treating cells with H₂O₂ for 18 h), HW+H₂O₂ group (culturing cells using culture media containing HW for 72 h in combination with H₂O₂ for 18 h), dexamethasone+ H₂O₂ group (treating cells using dexamethasone 1 μmol/L for 72 h in combination with H₂O₂ for 18 h). After culturing for 72 h in each group, the fluorescence intensity of reactive oxygen species (ROS) was measured by flow cytometry, and the contents of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected by ELISA. Results We successfully cultured orbital fibroblasts derived from orbital adipose connective tissues of TAO patients. The higher concentration of H₂O₂, the greater inhibition effect on the proliferation ability of the orbital fibroblasts from TAO patients, and we finally chose 100 μmol/L H₂O₂. After 72 h of cell culture, the contents of MDA, SOD, and GSHPx and the fluorescence intensity of ROS were (1.63±0.29), (5.06±0.24), (3.94±0.29), and (2.34±0.24) nmol/mL, (10.51±0.32), (2.41±0.23), (5.58±0.29), and (7.98±0.15) U/mL, (107.79±1.06), (21.07±0.92), (49.19±6.75), and (76.33±4.70) U/mL and 18 275.82±521.05, 92 524.81±2 097.01, 54 460.87±572.64, and 35 961.37±540.61 in the blank, H₂O₂, HW+ H₂O₂ and dexamethasone+H₂O₂ groups, respectively. Statistic analysis results showed that both HW and dexamethasone could significantly inhibit oxidative stress induced by H₂O₂ in orbital fibroblasts (all P<0.01), and the inhibitory effects of dexamethasone were significantly more obvious than those of HW (all P<0.01). Conclusion HW may be a treatment option for TAO through anti-oxidant stress.