Regulatory T cells (Tregs) are essential for pregnancy tolerance, and the peri-conception phase is critical for their generation at the outset of pregnancy(1). Treg insufficiency causes implantation failure in mice and is linked with infertility and gestational disorders in women. The thymic Treg (tTreg) compartment has not previously been evaluated in pregnancy tolerance. To investigate how tTreg as well as peripheral Tregs (pTregs) respond to male partner seminal fluid, FOXP3+CD4+Tregs were examined in the uterus and uterus-draining lymph nodes (dLN) in virgin estrus C57Bl/6 mice, and on 3.5 days post-coitum (dpc) after mating with Balb/c males.Flow cytometric analysis showed that mating elicited 5-fold increases in uterine Tregs accompanied by extensive Treg proliferation in the dLN. These cells comprised 70% tTregs identified by neuropilin-1 (NRP1) expression,and 30% NRP1- pTregs. tTregs exhibited a greater numerical increase and higher induction of proliferation marker Ki67 and suppressive competence markers FOXP3 and CTLA4 than pTregs. Analysis using the unbiased machine learning algorithm, t-stochastic neighbour embedding (tSNE) confirmed phenotypically distinct tTreg and pTreg clusters with dominant responsiveness to seminal fluid in the tTreg cluster. Bisulphite sequencing of FACS-sorted CD4+ T cells revealed increased demethylation of the Foxp3 locus Treg-specific demethylation region (TSDR) in tTregs but not pTregs after mating. These data show tTreg and pTregs both respond to seminal fluid priming, but greater phenotype attenuation occurs in tTregs, and the Foxp3epigenetic signature is uniquely increased in tTregs. We conclude that tTregs are sensitive to local regulation by seminal fluid. The tTreg compartment therefore warrants evaluation for potential to influence fertility and pregnancy health in women.