Significance The proteasome is responsible for the homeostasis of intracellular proteins. Here, we describe structural and functional aspects of a poorly characterized proteasome subtype found exclusively in germ cells. The spermatoproteasome was recently shown to be essential for spermatogenesis, a process requiring intense proteolysis. It differs from the constitutive proteasome by only one subunit, α4s, a subunit that replaces its α4 ubiquitous counterpart. In this work, we show how the shift from α4 to α4s regulates proteasome composition, dynamics, interactome, and activity. We reveal a regulation process more complex than previously suggested, which provides the basis for structural and functional studies of the spermatoproteasome.