Aberrant glycosylation is a featured characteristic of cancer and plays a role in cancer pathology; thus an understanding of thecompositions and functions of glycans is critical for discovering diagnostic biomarkers and therapeutic targets for cancer. In thisstudy, we used MALDI-TOF-MS analysis to determine theO-glycan profiles of prostate cancer cells metastasized to bone (PC-3), brain (DU145), lymph node (LNCaP), and vertebra (VCaP) in comparison to immortalized RWPE-1 cells derived fromnormal prostatic tissue. Prostate cancer (CaP) cells exhibited an elevation of simple/shortO-glycans, with a reduction of complexO-glycans, increasedO-glycan sialylation and decreased fucosylation. Core 1 sialylation was increased dramatically in all CaPcells, and especially in PC-3 cells. The expression of Neu5Acα2-3Galβ1-3GalNAc- (sialyl-3T antigen) which is the product ofα2,3-sialyltransferase-I (ST3Gal-I) was substantially increased. We therefore focused on exploring the possible function ofST3Gal-I in PC-3 cells. ST3Gal-I silencing studies showed that ST3Gal-I was associated with PC-3 cell proliferation, migrationand apoptosis. Furtherin vivostudies demonstrated that down regulation of ST3Gal-I reduced the tumor size in xenograft mousemodel, indicating that sialyl-3T can serve as a biomarker for metastatic prostate cancer prognosis, and that ST3Gal-I could be atarget for therapeutic intervention in cancer treatment.