Objective: Thyroid-associated ophthalmopathy (TAO) is an autoimmune disorder involving the orbital tissue. This study aimed to understand the role of regulatory T cells (Tregs) in TAO during 12-week systemic glucocorticoid (GC) treatment. Methods: Thirty-two moderate severe TAO patients with a clinical activity score (CAS) >= 3/7 or with prolonged T2 relaxation time (T2RT) on at least one side of extraocular muscle (EOM) were enrolled. The percentage of the peripheral CD4+CD25(high)CD127(-/low) Tregs was analyzed using flow cytometry before and after the GC treatment. The activity and severity of TAO, T2RT, and the clinical outcomes after the GC treatment were assessed. Their correlation with the peripheral Tregs was investigated. Results: There was no significant association between the baseline Treg fraction and the activity and severity of TAO or the treatment response. A significant reduction of Tregs was observed after the GC therapy merely in patients without any clinical improvement. Conclusion: Treg reduction after systemic GC therapy is indicative of a poor therapeutic response. Accordingly, dynamic alterations of Tregs could help to evaluate the effectiveness of the GC treatment.