Sperm storage in the female reproductive tract after mating and before ovulation is a reproductive strategy used by many species. When insemination and ovulation are poorly synchronized, the formation and maintenance of a functional sperm reservoir improves the possibility of fertilization. In mammals, the oviduct regulates sperm functions, such as Ca2+ influx and processes associated with sperm maturation, collectively known as capacitation. A fraction of the stored sperm is released by unknown mechanisms and moves to the site of fertilization. There is an empirical association between the hormonal milieu in the oviduct and sperm detachment; therefore, we tested directly the ability of progesterone to induce sperm release from oviduct cell aggregates. Sperm were allowed to bind to oviduct cells or an immobilized oviduct glycan and then challenged with progesterone, which stimulated the release of 48% of sperm from oviduct cells or 68% of sperm from an immobilized oviduct glycan. The effect of progesterone on sperm release was specific; pregnenolone and 17α-OH-progesterone did not affect sperm release. Ca2+ influx into sperm is associated with capacitation and development of hyperactivated motility. Progesterone increased sperm intracellular Ca2+, which was abrogated by blocking the sperm–specific Ca2+ channel CatSper with NNC 055-0396. NNC 055-0396 also blocked the progesterone-induced sperm release from oviduct cells or immobilized glycan. An inhibitor of the non-genomic progesterone receptor that activates CatSper similarly blocked sperm release. This is the first report indicating that release of sperm from the sperm reservoir is induced by progesterone action through CatSper channels.