Transient Receptor Potential Vanilloid sub-type 4 (TRPV4) is a non-selective cationic channel involved in regulation of temperature, osmolality and different ligand-dependent Ca2+-influx. Recently, we have demonstrated that TRPV4 is conserved in all vertebrates. Now we demonstrate that TRPV4 is endogenously expressed in all vertebrate sperm cells ranging from fish to mammals. In human sperm, TRPV4 is present as N-glycosylated protein and its activation induces Ca2+-influx. Its expression and localization differs in swim-up and swim-down cells suggesting that TRPV4 is an important determining factor for sperm motility. We demonstrate that pharmacological activation or inhibition of TRPV4 regulates Ca2+-wave propagation from head to tail. Such findings may have wide application in male fertility–infertility, contraception and conservation of endangered species as well.