Human semen, consisting of spermatozoa and seminal plasma, represents a special clinical sample type in human body fluid. Protein glycosylation in semen plays key roles in spermatogenesis, maturation, capacitation, sperm–egg recognition, motility of sperm and fertilization. In this study, we profiled the most comprehensive O-glycoproteome map of human semen using our recently presented glycoproteomics based on two complementary fragmentation methods (GlycoTCFM). We showed that semen contains a lot of novel and distinctive O-glycoproteins, which are mostly located in extracellular region and membrane, enriched in the biological processes of cell adhesion and angiogenesis, and mainly involved in multiple biological functions including extracellular matrix structural constituent and binding.