The 26S proteasome is a multi-subunit protease specifically targeting ubiquitinated proteins. A consensus has emerged from studies by multiple laboratories on the role of sperm-borne proteasomes in human, mouse, pig, bovine, ascidian and echinoderm fertilization. Major findings from the studies in various mammalian and non-mammalian fertilization systems are (1) proteasomes are present in the mammalian sperm acrosome and on the acrosomal surface; (2) ubiquitinated proteins are present on the mammalian, ascidian and echinoderm egg coat; (3) proteasomal proteolytic and ubiquitin-deconjugating (deubiquitinating) activities can be detected in viable, motile mammalian spermatozoa; (4) proteasomes remain associated with the sperm head following ZP-induced acrosomal exocytosis; (5) inhibition of ubiquitination and proteasomal proteolysis blocks fertilization in mammals, ascidians and echinoderms; (6) inhibition of proteasomal proteolysis alters the course of mammalian sperm capacitation and acrosomal exocytosis induced by sperm binding to the egg coat, zona pellucida (ZP); (7) depletion of the sperm surface-associated ATP blocks porcine and echinoderm fertilization, most likely by affecting the integrity of sperm proteasomes, of which several subunits are ATPases; (8) inhibition of proteasomal proteolysis blocks sperm-ZP penetration, but does not alter the rate of sperm-ZP binding in mammals, and (9) experimental modification of sperm-associated deubiquitinating activities shifts the balance of monospermic fertilization to polyspermic fertilization in vitro. Altogether, these studies provide evidence for the involvement of the 26S proteasome in multiple steps of animal and human fertilization, offering a novel model of sperm-egg coat interactions, and identifying a range of potential new sperm quality markers and contraceptive targets.