In mammals, the oviduct retains sperm forming a reservoir from which they are released in synchrony with ovulation. However, the mechanisms underlying sperm release are unclear. Herein, we first examined in greater detail the release of sperm from the oviduct reservoir by sex steroids, and second, if the ubiquitin-proteasome system (UPS) mediates this release in vitro. Sperm were allowed to bind to oviductal cells or immobilized oviduct glycans, either bi-SiaLN or a suLeX, and then challenged with steroids in the presence or absence of proteasome inhibitors. Previously, we have demonstrated that progesterone-induced sperm release from oviduct cells and immobilized glycans in a steroid-specific manner. Herein we found that release of sperm from an immobilized oviduct glycan, a 6-sialylated branched structure, and from immobilized fibronectin was inhibited by the CatSper blocker NNC 055-0396, akin to the previously reported ability of NNC 055-0396 to inhibit sperm release from another oviduct glycan, sulfated Lewis X trisaccharide. Thus, CatSper may be required for release from a variety of adhesion systems. One possible mechanism for sperm release is that glycan receptors on sperm are degraded by proteasomes or shed from the sperm surface by proteasomal degradation. Accordingly, the inhibition of proteasomal degradation blocked sperm release from oviduct cell aggregates and both immobilized oviduct glycans as well as fibronectin. In summary, progesterone-induced sperm release required both active CatSper channels and proteasomal degradation, suggesting that hyperactivation and proteolysis are vital parts of the mechanism by which sperm move from the oviduct reservoir to the site of fertilization.