The MST1 serine-threonine kinase, a component of the RASSF1-LATS tumor suppressor network, is involved in cell proliferation and apoptosis and has been implicated in cancer. However, the physiologic role of MST1 in prostate cancer (PCa) is not well understood. Here, we investigated the possibility of a biochemical and functional link between androgen receptor (AR) and MST1 signaling. We showed that MST1 forms a protein complex with AR and antagonizes AR transcriptional activity as shown by coimmunoprecipitation (co-IP), promoter reporter analysis, and molecular genetic methods. In vitro kinase and site-specific mutagenesis approaches indicate that MST1 is a potent AR kinase; however, the kinase activity of MST1 and its proapoptotic functions were shown not to be involved in inhibition of AR. MST1 was also found in AR-chromatin complexes, and enforced expression of MST1 reduced the binding of AR to a well-characterized, androgen-responsive region within the prostate-specific antigen promoter. MST1 suppressed PCa cell growth in vitro and tumor growth in mice. Because MST1 is also involved in regulating the AKT1 pathway, this kinase may be an important new link between androgenic and growth factor signaling and a novel therapeutic target in PCa. Cancer Res; 71(12); 4303-13. (C)2011 AACR.