Protamines (PRMs) and transition nuclear proteins (TNPs) are two key classes of sperm nuclear basic proteins that regulate chromatin reorganization and condensation in the spermatozoon head, playing crucial roles in mammalian spermatogenesis. In scrotal mammals, such as humans, cryptorchidism, the failure of the testes to descend into the scrotal sac is generally associated with higher rates of defective spermatozoon quality and function. However, ascrotal mammals, such as cetaceans, with naturally undescended testes, produce normal spermatozoa similar to their scrotal counterparts. This study investigates the evolutionary pattern and functional changes in PRMs and TNPs to explore the potential molecular mechanisms underlying spermatogenesis in naturally ascrotal mammals. Although we found a conserved genomic arrangement for PRM and TNP genes across mammals, the coevolutionary loss of intact PRM2 and TNP2 was observed in several species, correlating significantly with diverse testicular positions. Notably, in cetaceans, which lack intact PRM2 and TNP2, we detected enhanced thermostability and DNA binding in PRM1, along with superior DNA repair capability in TNP1. These findings suggest that gene loss of PRM2 and TNP2, combined with functional enhancements in PRM1 and TNP1 proteins, evolved in response to physiological challenges posed by natural cryptorchidism in most ascrotal lineages. This evolutionary strategy enhances chromatin condensation efficiency and promotes DNA repair during spermatogenesis in natural cryptorchid mammals, supporting the Black Queen Hypothesis.