Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.