Prostate cancer is the most commonly diagnosed form of cancer and the second leading cancer-related death among men in the Western civilization. Since no effective therapy exists for this tumor after progression beyond resectable boundaries, there is an urgent need for new treatment strategies. Prostate specific membrane antigen (PSMA) represents an excellent target on prostate cancer cells, and therefore specific immunotherapy may be a novel therapeutic option for the management of this tumor. We constructed a fully recombinant immunotoxin (A5-PE40) from a single-chain antibody fragment (scFv) against cell-adherent PSMA and a truncated form of Pseudomonas exotoxin A (PE40) lacking its natural binding domain Ia. The scFv A5 was obtained from a mAb elicited with native PSMA by phage display technology and direct selection on cells carrying the antigen. The bacterially expressed and purified immunotoxin A5-PE40 specifically binds to PSMA-positive prostate cancer cells and induces a 50% reduction of viability (IC50) at a concentration of 20 pM, while PSMA-negative cells remain unaffected. Due to its high and specific toxicity this recombinant immunotoxin is a promising candidate for therapeutic applications in patients with prostate cancer.