To ensure genome stability across generations, an egg must be fertilized by only a single sperm. However, how sperm actively contribute to preventing polyspermy has remained unknown. Here, we demonstrate that testis-specific 20S proteasome core particles (CPs) are pre-stored within the sperm heads of both mice and humans. Upon fertilization, these testis-specific 20S CPs assemble with oocyte-derived 19S regulatory particles (RPs), forming chimeric proteasomes that efficiently degrade Fetuin B, which is ubiquitinated by the E3 ligase MARCH3. The clearance of Fetuin B triggers ZP2 cleavage and subsequent zona pellucida hardening, thereby blocking additional sperm entry. The newly assembled chimeric proteasome in the zygote represents a novel strategy to prevent polyspermy during fertilization in mammals.