Castration-resistant prostate cancer (CRPC) is a malignant tumor that is resistant to androgen deprivation therapy. Treatments for CRPC are limited, and no diagnostic markers are currently available. O-glycans are known to play an important role in cell proliferation, migration, invasion, and metastasis of cancer cells. However, the differences in the O-glycan expression profiles for normal prostate cancer (PCa) cells compared to CRPC cells have not yet been investigated. In this study, the saccharide primer method was employed to analyze the O-glycans expressed in CRPC cells. Expression levels of core 4-type O-glycans were significantly increased in CRPC cells. Furthermore, the expression level of N-Acetylglucosaminyltransferase 3 (GCNT3), a core 4-type O-glycan synthase gene, was increased in CRPC cells. The expression of core 4-type O-glycans and GCNT3 was presumed to be regulated by androgen deprivation. GCNT3 knockdown induced cell migration and epithelial-mesenchymal transition (EMT). These observations elucidate the mechanism of acquisition of castration resistance in PCa and offer new possibilities for the development of diagnostic markers and therapeutic targets in the treatment of PCa.