The deposition of seminal fluid into the female reproductive tract at coitus elicits an inflammation-like response characterised by induction of cytokines and chemokines and extensive leukocyte recruitment. In mice, this response is important for preparing the female reproductive tract for successful embryo implantation through promoting immune tolerance towards male allo-antigens. Seminal plasma (SP) has been considered the sole signalling component of semen, however recent mouse studies have shown spermatozoa also contribute to eliciting the female immune response. Whether spermatozoa also contribute to seminal fluid signalling in men remains unclear. In this study, we investigated the capacity of human spermatozoa to interact with immortalised ectocervical epithelial (Ect1) cells to induce cytokine and chemokine synthesis using an in vitro seminal fluid signalling model. We observed that washed spermatozoa - as well as whole semen and SP – acted to elicit Ect1 cell production of colony stimulating factor (CSF) 2 (mean of 3-fold increase), CSF3 (5-fold increase), interleukin (IL) 1A (2.3-fold increase), IL6 (1.7-fold increase), CC chemokine ligand (CCL) 2 (1.6-fold increase), CCL3 (1.9-fold increase), and chemokine (C-X-C motif) ligand 8 (CXCL8, 6.6-fold increase). Considerable variation in the profile of cytokines and chemokines induced by sperm from Ect1 cells existed between individual men. Live-cell confocal imaging experiments demonstrated active, reversible binding of subsets of spermatozoa to Ect1 cells. In addition, scanning and transmission electron microscopy showed evidence of close attachment with microvillus structures protruding from the Ect1 cell surface to form intimate associations with the head of the spermatozoa, and in some instances, potential engulfment by Ect1 cells. Currently, the underlying mechanisms and biological significance of sperm-mediated signalling in cervical epithelial cells is unknown. This preliminary data suggests a previously unknown biological activity of spermatozoa in eliciting the female immune response to seminal fluid.