The growth factor proepithelin has recently emerged as an important regulator of transformation in several physiological and pathological systems. In this study, we determined the biological roles of proepithelin in prostate cancer cells using purified human recombinant proepithelin as well as proepithelin-depletion strategies. Proepithelin promoted the migration of androgen-dependent and -independent human prostate cancer cells; androgen-independent DU145 cells were the more responsive. In these cells, proepithelin additionally stimulated wound closure, invasion, and promotion of cell growth in vitro. These effects required the activation of both the Akt and mitogen-activated protein kinase pathways. We have analyzed proepithelin expression levels in different available prostate cancer microarray studies using the Oncomine database and found a statistically significant increase in proepithelin mRNA expression levels in prostate cancers compared with nonneoplastic controls. Notably, depletion of endogenous proepithelin by siRNA and antisense strategies impaired the ability of DU145 cells to grow and migrate after serum withdrawal and inhibited anchorage-independent growth. Our results provide the first evidence for a role of proepithelin in stimulating the migration, invasion, proliferation, and anchorage-independent growth of prostate cancer cells. This study supports the hypothesis that proepithelin may play a critical role as an autocrine growth factor in the establishment and initial progression of prostate cancer. Furthermore, proepithelin may prove to be a useful clinical marker for the diagnosis of prostate tumors.