CD4+ CD25+ regulatory T (Treg) cells play an important role in the control of the immune system by suppressing the proliferation of effector cells, thereby preventing autoreactive, unnecessary or inconvenient responses. Recently, it has been shown that the number of Treg cells increases during pregnancy, a period with high serum levels of female sex hormones. Oestrogen replacement therapy has been reported to alleviate the symptoms of autoimmune diseases, yet the cellular and molecular mechanisms involved are not fully understood. Here, we show that physiological doses of oestradiol (E2) found during pregnancy, combined with activation through CD3/CD28 engagement, promoted the proliferation of Treg cells without altering their suppressive phenotype. Enhanced suppression was detected when Treg cells were pretreated with the hormone as well as when both cell subpopulations (Treg and T effector) were exposed to E2 throughout the experiment. Together, these data suggest that when combined with an activating stimulus, E2 can modulate the function of human Treg cells by regulating their numbers, and highlight a potential use of E2, or its analogs, to manipulate Treg function.