Expression of HEPSIN, a type II transmembrane serine protease in prostate cancer (CaP), has been highlighted by several studies analyzing CaP-specific gene expression alterations by cDNA microarray. Evaluations of the biological functions of HEPSIN in CaP cells are warranted for better assessment of its utility as a biomarker and/or therapeutic target. In stable clones of PC-3/HEPSIN transfectants, there was a dramatic reduction in the cell growth, cell invasion, and soft agar colony formation. A higher proportion of PC-3/HEPSIN cells were in the G(2)-M phase of the cell cycle, and there was also an increase in the cell population undergoing apoptosis. Preliminary analysis of HEPSIN transfections into LNCaP and DU145 cells further revealed cell growth-inhibitory effects. These results underscore that exogenous HEPSIN expression negatively regulates cell growth in metastatic CaP cell lines. Although the cause of the biological consequence of HEPSIN overexpression in primary CaP remains to be determined, the negative cell growth-regulatory effects of HEPSIN in metastatic CaP cells reported here have unraveled possible cellular and molecular mechanisms underlying observations that link decreased/loss of HEPSIN expression with poor prognosis of CaP.