Prostate-specific antigen (PSA) two-dimensional electrophoresis (2-DE) subforms (F1–F5) have been described to be altered in prostate cancer (PCa) compared to benign prostatic hyperplasia (BPH). To understand their molecular differences, characterization of these subforms from PCa serum and seminal plasma, namely, at the glycan level, was performed. PSA 2-DE subforms from two serum PCa samples and seminal plasma were analyzed by N-glycan sequencing using high-performance liquid chromatography (HPLC) combined with exoglycosidase array digestions and by mass spectrometry. F1, F2, and F3 subforms showed the same N-glycan pattern, which contained higher levels of sialic acid than the F4 subform, whereas the F5 subform was unglycosylated. When comparing PSA subforms from PCa with seminal plasma, a decrease in sialylation was observed. Furthermore, the analysis of F3, the more abundant PSA subform, showed a higher proportion of alpha 2–3 sialic acid and a decrease in core fucosylated glycans in the PCa sample. These N-glycan changes in PCa PSA subforms highlight the importance of glycosylation as an indicator of PCa disease