We have previously identified the Mitostatin gene, localized at 12q24.1, in the process of screening for growth-arrested genes induced by the leucine-rich proteoglycan decorin. Decorin is a member of the small leucine-rich proteoglycan gene family that has recently become a focus in several areas of cancer research. This soluble protein is involved in a number of cellular processes including matrix assembly, fibrillogenesis, and the control of cell proliferation. Decorin has been shown to inhibit migration, invasion, and tumorigenicity of a wide variety of transformed cells. CBIQ Moreover, decorin induces apoptosis through the activation of caspase-3. Hence, it is plausible, that decorin-induced proteins could be effectors of the tumor suppressive action of this proteoglycan. We have previously shown that Mitostatin is ubiquitously expressed in normal human tissues. However, its protein levels are markedly attenuated in advanced stages of primary mammary and urothelial neoplasms. We further demonstrated that Mitostatin over-expression negatively affects cell growth and induces cell death in bladder cancer cell lines, suggesting that Mitostatin could behave as a classical tumor suppressor gene in other forms of malignancy. In the present study we utilized three widely used prostate carcinoma cell lines, namely, PC3 and DU145 castrationresistant cells, and LNCaP androgen-dependent cells, and utilized transgenic and immunological strategy to pinpoint the function of Mitostatin in these cells. Our results confirmed our prediction of Mitostatin belonging to the tumor suppressor gene family AS-136A insofar as overexpression of Mitostatin inhibited colony formation, whereas suppression of endogenous Mitostatin caused the opposite effects. Cell migration and invasion are fundamental components of tumor cell metastases. As increased cell migration and invasion are hallmarks of the metastatic phenotype, and thus a measure of aggressiveness, the current study provides results that implicate Mitostatin as an important protein for determining an aggressive cellular phenotype. Indeed, Mitostatin over-expressing clones showed a significant decrease in motility, and vice-versa by downregulating endogenous Mitostatin with either antisense or siRNA strategies we triggered the opposite result.