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. Both sperm and seminal plasma act as signalling agents. In seminal plasma TGFβ has been identified as a signalling molecule, but sperm signalling molecules remain unknown. This study aimed to identify sperm signalling molecules using Affymetrix microarray and bioinformatics strategies. Gene expression profiles from mouse endometrium of unmated estrus CBAF1 females or females mated with either seminal vesicle deficient and vasectomised (SVX/VAS), vasectomised or intact males was examined by microarray and quantitative PCR (qPCR). Cytokine protein profiles were assayed by Luminex. A total of 139 genes were differentially regulated (>1.5-fold change, p<0.01 FDR corrected) in females mated with intact compared to vasectomised males, many of which were associated with immune signalling pathways. Bioinformatic analysis of the differentially regulated genes to identify candidate signalling molecules in sperm identified the TLR4 signalling pathway as a major upstream regulator. Key peri-conception cytokines that were predicted to be activated by TLR4, including GCSF, MIP2, IL6 and TNF were also induced following sperm exposure at the level of mRNA and protein (p<0.05). To examine the putative role of TLR4 in cytokine induction in the female tract, mouse uterine epithelial cells were cultured in vitro in the presence of the TLR4 agonist LPS and supernatants were assayed by Luminex. TLR4 ligation significantly induced GCSF, MIP2, IL6 and TNF (p<0.05). The requirement for TLR4 ligation in cytokine induction was confirmed using Tlr4 null mutant mice, where seminal fluid failed to induce endometrial Csf3, Cxcl2, Il6, and Tnf expression. This study provides evidence that TLR4 ligation following sperm exposure contributes to modulation of the periconception immune environment. Current studies are identifying and characterising the TLR4 ligands carried by sperm that elicit these changes.