Protein O-glycosylation plays critical roles in sperm formation and maturation. However, detailed knowledge on the mechanisms involved is limited due to lacking characterization of O-glycoproteome of testicular germ cells. Here, we performed a systematic analysis of site-specific O-glycosylation in mouse testis, and established an O-glycoproteome map with 349 O-glycoproteins and 799 unambiguous O-glycosites. Moreover, we comprehensively investigated the distribution properties of O-glycosylation in testis and identified a region near the N-terminal of peptidase S1 domain that is susceptible to O-glycosylation. Interestingly, we found dynamic changes with an increase Tn and a decrease T structure from early to mature developmental stages. Notably, the importance of O-glycosylation was supported by its effects on the stability, cleavage, and interaction of acrosomal proteins. Collectively, these data illustrate the global properties of O-glycosylation in testis, providing insights and resources for future functional studies targeting O-glycosylation dysregulation in male infertility.