MUC1 mucin is up-regulated and aberrantly glycosylated in many human epithelial carcinomas. Over-expression of MUC1 has also been implicated in prostate cancer, but neither the role of MUC1 in the cancer progression nor the mucin O-glycosylation has been fully elucidated. In this study, we characterized the O-glycans on MUC1 when over-expressed in the human prostate cancer cell line C4-2B(4). We found that the main O-glycan consisted of the neutral core 2 oligosaccharide Galβ3(Galβ3/4GlcNAcβ6)GalNAc-ol, with minor components being fucosylated and sialylated variants of the same core 2 oligosaccharide. Small amounts of the shorter core 1 O-glycans were also detected.We then used the MUC1 over-expressing cell lines to study the effects of MUC1 on prostate cancer cell behavior. The results demonstrate that over-expression of MUC1 did not affect the cells' proliferation, but led to a decreased adhesion to the extracellular matrix components fibronectin and collagen I in vitro. When inoculated in BALB/c nude mice, C4-2B(4) cells expressing MUC1 showed a tendency to form fewer tumors than wt cells and the tumors also grew more slowly, but there was a large variation between different tumors. These findings suggest that MUC1 may not have the same cancer-promoting effect in prostate cancer cells that is commonly seen in other epithelial cancers such as breast, colon, and pancreatic cancer.