Female regulatory T (Treg) cells are activated by seminal fluid factors following coitus. These activated Treg cells exert immune-suppressive actions which are essential to prevent immune rejection of the fetus. Reduced abundance and/or impaired function of Treg cells are evident in women experiencing pregnancy pathologies including recurrent miscarriage and preeclampsia. It is well established that long term sexual co-habitation and seminal fluid exposure, in a partner-specific manner, reduces the risk of pathologies associated with immune dysregulation such as preeclampsia and in utero growth restriction. Thus we hypothesised that repeated priming with seminal fluid can progressively expand the Treg cell pool. To test this female C57Bl/6 (B6) mice were mated once, twice or four times to B6 or Balb/C males. To allow the impact of seminal fluid to be delineated, mated female mice where administered the progesterone receptor antagonist RU486 to prevent pregnancy progression, before re-mating at least 7 days later. Uterus and para-aortic lymph nodes (PALN) were recovered at day 3.5 post-coitum after the final mating and Treg populations were assessed by flow cytometry, immunohistochemistry and in vitro suppression assays. Treg cell numbers in the uterus and PALN increased with each mating event. This expansion was dependent upon exposure to male seminal fluid factors, including paternal MHC antigen, as expansion was greater in allogeneic matings. Higher proportions of Treg cells isolated from mice mated four times expressed the proliferation marker Ki67 and suppression marker CTLA4, and had a greater capacity to suppress T cell responses as assessed by in vitro suppression assays. These data provide evidence that repeated seminal fluid contact acts to increase the number and suppressive capacity of the maternal Treg cell pool during early pregnancy. This data provides a mechanistic explanation for the link between duration of sexual co-habitation and protection from immune-based pregnancy complications in women.