The semi-allogeneic fetus is a major challenge to the mother’s immune system during pregnancy. Amongst the factors that contribute to maternal immune tolerance, regulatory T (Treg) cells play a pivotal role. We have previously demonstrated that exposure to seminal fluid increases Treg cell populations in the uterus. In this study, we aimed to determine whether repeated exposure would further expand the Treg cell pool. Female C57Bl/6 (B6) female mice were mated either once or four times to B6 males (syngeneic) or Balb/c males (allogeneic), using RU486 administration to prevent embryo implantation and pregnancy. As a mating control, females were mated to seminal vesicle deficient and vasectomised (SVX/VAS) males. Uterine tissue and lymph nodes were harvested at day 3.5 post-coitum of the final mating. Using immunohistochemistry and flow cytometry for the unique marker Treg cell transcription factor Foxp3, we demonstrated the Treg cell pool was expanded in both the uterus and the para-aortic lymph nodes after repeated exposure to allogeneic seminal fluid at day 3.5 post-coitum. Seminal fluid, as opposed to neuroendocrine or physical responses to mating were necessary for Treg cell pool expansion, since mating to SVX/VAS males failed to elicit the increase of Treg cells. However, no increase in the Treg cell population was observed after repeated syngeneic matings, indicating the significance of male transplantation antigens in expanding the Treg cell pool. Collectively, these data provide evidence that repeated exposure to seminal fluid acts to expand the Treg cell pool in the uterus during early pregnancy, and that active factors in seminal fluid are necessary to elicit the Treg cell response.