Successful growth of the semi-allogeneic fetus during mammalian pregnancy is dependent upon the development of maternal immune tolerance to fetus-derived foreign antigens. The activation and expansion of inducible regulatory T (Treg) cell populations is essential for this process. Factors in seminal fluid, delivered to the female at the time of insemination, are required for optimal Treg cell expansion. Work in a number of different species, including human, shows that repeated, long-term exposure to seminal fluid protects from immune-related pregnancy disorders leading to healthier progeny. Here, we aimed to determine whether repeated exposure to seminal fluid acts to expand the Treg cell pool. Female C57Bl/6 mice were mated once or four times to syngeneic or allogeneic (BALB/c) males, with pregnancies prevented by RU486 administration. The uterus-draining para-aortic lymph nodes (PALN) and uterus were collected after the final mating and analysed by flow cytometry and immunohistochemistry, respectively. Four allogeneic matings increased Treg cell numbers in the PALN and uterus compared to a single allogeneic mating. Four syngeneic matings did not lead to the same increase suggesting that MHC alloantigens play a role in Treg cell expansion. In additional experiments, females mated four times to males that had been surgically altered to lack sperm (vasectomised) or seminal plasma (seminal vesicle removed) or both, failed to generate a strong Treg cell response, suggesting that seminal fluid factors were critical for optimal Treg cell expansion. Using immunohistochemistry the macrophage markers F4/80 and MHC II were analysed in the uterus. While multiple matings did not change the frequency of macrophages within the uterus, MHC IIwas increased. Collectively, these data suggest that a greater number of activated antigen presenting cells in the uterus contributes to progressive expansion of the Treg cells pool in mice repeatedly exposed to allogeneic seminal fluid. This may provide a mechanistic explanation for the protection which pregnant women receive from long-term seminal fluid exposure and sexual cohabitation with their conceiving partner.