O-GlcNAc is an O-linked ?-N-acetylglucosamine moiety attached to the side-chain hydroxyl of a serine or threonine residue in numerous cytoplasmic and nuclear proteins. In this study, we detected the level of O-GlcNAc in prostate, liver and pancreatic cancer tissues, and found that the global O-GlcNAc modification also known as O-GlcNAcylation, is specifically increased in prostate cancer tissues compared to corresponding adjacent tissues. In addition, we found that global O-GlcNAcylation is increased in prostate cancer cells and not in benign prostatic hyperplasia (BPH) epithelial cells. O-GlcNAc enhanced the anchorage-independent growth and the migratory/invasive ability of prostate cancer cells. More importantly, we provide here, for the first time to the best of our knowledge, direct evidence that increased O-GlcNAcylation induces malignant transformation of nontumorigenic (BPH) cells. Furthermore, our study suggested that inhibiting the formation of the E-cadherin/catenin/cytoskeleton complex may underly the O-GlcNAc-induced prostate cancer progression. Overall, these findings indicated that O-GlcNAcylation is increased in prostate, but not in liver and pancreatic cancer tissues, and that O-GlcNAc can enhance the malignancy of prostate cancer cells.