Oestrogen controls Foxp3 expression in regulatory T cells (T_reg cells) via a mechanism thought to involve oestrogen receptor alpha (ERα), but the molecular basis and functional impact of ERα signalling in T_reg cells remain unclear. We report that ERα ligand oestradiol (E2) is significantly increased in human cervical cancer (CxCa) tissues and tumour-infiltrating T_reg cells (CD4⁺CD25^hiCD127^low), whereas blocking ERα with the antagonist ICI 182,780 abolishes FOXP3 expression and impairs the function of CxCa infiltrating T_reg cells. Using a novel approach of co-immunoprecipitation with antibodies to E2 for capture, we identified binding of E2:ERα complexes to FOXP3 protein in CxCa-derived T_reg cells. Chromatin immunoprecipitation analyses of male blood T_reg cells revealed ERα occupancy at the FOXP3 promoter and conserved non-coding DNA elements 2 and 3. Accordingly, computational analyses of the enriched regions uncovered eight putative oestrogen response elements predicted to form a loop that can activate the FOXP3 promoter. Together, these data suggest that E2-mediated ERα signalling is critical for the sustenance of FOXP3 expression and T_reg cell function in human CxCa via direct interaction of ERα with FOXP3 promoter. Overall, our work gives a molecular insight into ERα signalling and highlights a fundamental role of E2 in controlling human T_reg cell physiology.