The dysfunction of regulatory T cell (Treg) is associated with the pathogenesis of many immune diseases. The regiments used to re-establish Treg's function are currently unsatisfactory and need to be improved. The purpose of this study is to elucidate the synergistic effects of cortisol and endoplasmic reticulum (ER) stress on impairing regulatory T cell functions. In this study, blood samples were collected from patients with food allergy (FA). Immune cells were purified from blood specimens by flow cytometry. A mouse model of FA was established with ovalbumin as a specific antigen. We observed that serum cortisol levels of FA patients were negatively correlated with peripheral Treg counts. Overwhelmed ER stress status was detected in Tregs of FA patients. The antigen-specific immune response induced ER stress in Tregs, which was exacerbated by concurrent cortisol exposure. ER stress mediated the effects of cortisol on impairing the immune suppressive ability of Tregs. The expression of Rnf20 was observed in Tregs upon exposure to cortisol. Rnf20 reduced the expression of Foxp3 and transforming growth factor (TGF)-β in Tregs. Rnf20 inhibition re-established the immunosuppressive functions of Tregs obtained in patients with FA. The experimental FA in mice was attenuated by inhibition of Rnf20 in Tregs. In summary, specific immune response in synergy with cortisol to induce the expression of Rnf20 in Tregs. Rnf20 reduces the levels of Foxp3 and TGF-β to impair the immune suppressive function. Inhibition of Rnf20 can restore the immune suppressive ability of Tregs obtained from FA patients.© 2023 John Wiley & Sons Ltd.