Previous studies have identified a subclone cell line (PC-J) which was isolated from a metastatic human prostate cell line, PC-3. In vitro matrigel invasion assays and xenograft animal studies suggested that matriptase was a putative metastatic gene in human prostate carcinoma cells. Although low metastatic prostate tumor cells, LNCaP, also expressed high levels of matriptase mRNA, gelatin zymography indicated that LNCaP cells had extremely low matriptase activity. Further studies using RT-PCR and lectin blotting assays revealed that the expression of N-acetylglucosaminyltransferase V (MGAT5), a glycoprotein that stabilizes matriptase, was low in LNCaP cells compared to PC-3 and PC-J cells. The transient overexpression of MGAT5 significantly enhanced the activity of matriptase and the invasion ability in the LNCaP cells. Knock-down of MGAT5 in PC-3 cells attenuated the metastatic ability of the cells, as determined by the in vitro invasion assay and the xenograft animal studies. Matriptase and MGAT5 may play important role in the metastasis of prostate cancer.