In rodents, seminal fluid establishes maternal immune tolerance for embryo implantation and optimal placentation by inducing inflammatory changes and priming the immune response to paternal antigens. Coital exposure to seminal fluid also induced marked changes in gene expression, cytokine and chemokine production and subsequent leukocyte recruitment within cervical tissues. This study was undertaken to determine whether seminal fluid also altered gene expression profiles in human endometrial tissues in vivo, and in Ishikawa endometrial epithelial cells lines in vitro. With informed consent, women (n=4) with a past history of tubal ligation and regular menstrual cycles were recruited via community notices to this randomised, cross over design, pilot study. Volunteers who used hormonal or barrier contraception, had irregular cycles or did not have a consenting sexual partner were excluded. Urinary ovulation prediction kits identified the luteinising hormone (LH) surge. At the first positive LH test, each participant was randomised to either have unprotected intercourse or abstain in the next 24 hours in 2 different menstrual cycles, 2 months apart. Twelve hours after timed intercourse or abstinence, endometrial biopsies were collected by pipelle and immediately stored in RNAlater before RNA extraction. Affymetrix microarray and qPCR analyses identified immune-regulatory and tissue remodelling genes that were consistently differentially expressed when women had had intercourse (n=3). CXCL8 (3.8-fold), MMP12 (3.6-fold), IL1RN (1.6-fold), CXCL5 (1.5-fold) and TNFSF10/TRAIL (1.7-fold) were upregulated, whereas CXCL14 (3.3-fold) and CSF1 (1.4-fold) were downregulated. These changes were not seen in the fourth woman who abstained in both treatment cycles. In vitro, whole seminal fluid, seminal plasma and washed sperm elicited similar gene expression profiles in Ishikawa cells. Our study provides the first evidence that coital seminal fluid exposure may alter gene expression in the human endometrium. Human endometrial responses to seminal fluid exposure potentially contribute to embryo implantation and subsequent fetal development.