The fetus is antigenically distinct from the mother and therefore the maternal immune system must establish immunological tolerance towards the fetus to support pregnancy. Maternal tolerance is primarily mediated by a specialised subset of CD4+ T cells known as regulatory T (Treg) cells. An absence or reduced function of Treg cells during the peri-implantation period leads to pregnancy loss in mice and is implicated as a cause of infertility and pregnancy disorders in women. The pregnancy hormone, progesterone (P4), has potent immunosuppressant effects which act through the nuclear progesterone receptor (PR), which is expressed by Treg cells. We hypothesized that P4, through PR, regulates Treg cell abundance and phenotype in the hormone environment of early pregnancy. A hormone replacement model was used, whereby mice were ovariectomised (OVX) and treated with estrogen (E2)+P4. Using flow cytometry, an increase in the number of CD4+ T cells and CD4+CD25+ Foxp3+ Treg cells in the uterus-draining lymph nodes (LNs) and spleen was observed in mice treated with E2+P4 compared to control treatment groups. Additionally, female PR null mutant mice (PR-/-) and wild type mice (PR+/±) were OVX and treated with E2+P4. When compared with PR+/± mice, PR-/- mice had decreased frequencies of Treg cell in the uterus-draining LNs. To investigate the role of P4 in regulating Treg cell phenotype, splenocytes were isolated from mice and cultured under conditions to polarize differentiation towards TH1 or TH17 cells in the presence or absence of P4. When CD4+ T cell cytokine expression was subsequently measured using flow cytometry, P4 was found to repress IFNγ expression in Treg and T effector cells. Collectively, this work demonstrates that P4, potentially acting through PR, can regulate Treg cell abundance and cytokine production, which may be important in the establishment and maintenance of competent maternal tolerance during pregnancy.