Objective To study the role of deubiquitinating enzyme 16 ( Usp16 ) in spermatogenesis by establishing a mouse model of conditional Usp16 knockout in germ cells. Methods Vasa-cre transgenic mice were crossed with Usp16 transgenic mice (Usp16^{flox / flox}) to obtain germ cell-specific Usp16 knockout mice. To confirm that Usp16 had been conditionally inactivated in germ cells, genotyping by PCR and western blotting was performed to evaluate Usp16 expression. Initial characterization of the phenotype of conditional knockout (CKO) mice was then conducted by co-cage mating, computer-assisted sperm analysis, and HE staining. Results We confirmed that Usp16 expression was appropriate in the conditional knockout mouse. Compared with the wild type, the conception rate of female mice mated with CKO mice had decreased to only 7%, while no significant difference was observed in heterozygous knockout mice male mice. The sperm count of CKO mice was decreased by approximately 79%, motility was decreased by approximately 87%, and the rate of sperm deformity was as high as 83%. Multinucleated cells appeared in the seminiferous tubules of the testes and the number of precocious sperm in the epididymis had increased by approximately 76%. Conclusions Homozygous mutation of Usp16 in germ cells severely reduces sperm quality, thereby reducing male fertility.