N-linked glycosylation is an important type of post-translational modification. This modification plays important roles in many cellular functions including cell-cell and receptor-ligand interactions, immune response, apoptosis, and pathogenesis of many diseases. Seminal plasma is a mixture of secretions from several male accessory glands. Glycoproteins in seminal plasma can reflect the states of reproductive organs or diseases. Using isotope labeling and high-resolution mass spectrometry, we identified 720 N-glycosylated sites on 372 N-glycosylated proteins, corresponding to 365 genes. Analysis of variations among five individuals revealed similar compositions of N-glycosylated proteins in seminal plasma. Consistent composition of N-linked glycoproteins in human seminal plasma provides evidences that seminal plasma glycoproteins are of significant value to be candidates of biomarkers. Bioinformatics annotation of the identified seminal plasma glycoproteins showed that more than 43.0% of these glycoproteins are known secreted proteins according to UniProt. Such a high proportion of secreted glycoproteins in seminal plasma may play important roles in seminal plasma function such as making sure for sperm nutrition and function.