Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce pro-inflammatory cytokines and chemokines, which in turn initiate immunological adaptations required for pregnancy. Factors in the seminal plasma fraction including TGFβ family members have been identified as key signalling agents, but these don’t fully account for the female response. We hypothesised that seminal fluid contains novel signalling molecules in addition to TGFβ that activate immune signalling pathways, leading to immune tolerance. Gene expression was examined by microarray and qPCR in endometrium of unmated estrus CBAF1 female mice, or mice mated with either INTACT males, or males that were seminal vesicle deficient and vasectomised (SVX/VAS). Cytokines of interest were further analysed by Luminex to determine protein levels. Microarray and qPCR data confirmed that seminal fluid induces key peri-conception cytokines G-CSF, IL6, KC and LIF at the level of both mRNA and protein. Bioinformatic analysis of the differentially expressed genes identified a number of immune signalling pathways activated by seminal fluid. As well as TGFβ, TLR4 was identified as a key upstream regulator of these genes. To examine the role of TLR4 activation by seminal fluid, we utilised Luminex to examine the secretion of cytokines by the TLR4 agonist LPS, compared to TGFβ, in both primary mouse endometrial cells and human Ect1 ectocervical epithelial cells in-vitro. Both G-CSF and KC, which were not induced by TGFβ, were strongly induced following TLR4 ligation. LPS was present in human seminal fluid, but the concentration was insufficient to explain the observed cytokine increases. These studies are the first to provide evidence for TLR4 ligands contributing to the peri-conception immune environment. Activation of TLR4 signalling by microbial or endogenous components of seminal fluid may be a key pathway in generating the appropriate immune response required for the generation and maintenance of immune tolerance.