Immune adaptation to accommodate pregnancy requires sufficient T regulatory (Treg) cells in the endometrium to prevent maternal immune rejection during the critical peri-implantation period. We have previously demonstrated that exposure to seminal fluid from same male at coitus stimulated Treg cell proliferation and their recruitment into the uterus, and also further expanded Treg cell population in the uterus draining lymph nodes (PALNs) as well as uterus. We aimed to determine whether repeated exposure to seminal fluid acts to expand the Treg cell pool beyond that seen in a single mating, thereby providing greater immune tolerance and protection. Female C57Bl/6 mice were mated once, twice or four times to syngeneic (C57Bl/6) or allogeneic (BALB/c) males, with progression to pregnancy prevented by administration of the progesterone-signalling antagonist RU486. The uterus-draining para-aortic lymph nodes (PALN) and uterus were collected and analysed by flow cytometry and immunohistochemistry.In the PALN, Treg cell numbers progressively increase with each allogeneic mating, with inducible Treg cells having increased expression of the suppression marker CTLA4. Likewise, repeat seminal fluid exposures lead to an increase in uterine Treg cell numbers, with a higher expression of the signature transcription factor Foxp3, and increased CTLA4 expression.These responses were not seen in syngeneic matings confirming that MHC alloantigens play a key role in Treg cell expansion. Minor histocompatibility antigens did not significantly influence Treg cell expansion, as C57Bl/6 female mice mated to minor antigen disparate males (BALB/b) on four occasions did not have elevated Treg cell numbers. Four matings also enhanced the number of activated antigen presenting cells within the uterus. Taken together, multiple allogeneic seminal fluid exposure increases activated uterine APCs and Treg cell numbers, which may provide a mechanistic explanation for the reduced rates of pregnancy complications seen in women with long-term seminal fluid exposure to their conceiving partner.