Purpose: Overexpressed cytokine receptors are considered valid targets for new biologicals targeting prostate cancer. However, current reagents are limited in efficacy. Our goal was to determine the advantages of simultaneously targeting two established targets, epidermal growth factor receptor and interleukin-13 (IL-13) receptor, with a new bispecific cytotoxin in which both EGF and IL-13 cytokines were cloned onto the same single-chain molecule with truncated diphtheria toxin (DT390). Experimental Design: In vitro experiments measured the potency of bispecific DTEGF13 and compared its activity to its monospecific counterparts, DTEGF and DTIL13. We determined whether the presence of both cytokine ligands on the same molecule was responsible for its superior activity. In vivo, DTEGF13 was given i.t. to athymic nude mice with established PC-3 human prostate cancer tumor xenografts on their flanks. Results: In vitro, DTEGF13 was more potent than the monospecific cytotoxins against human prostate cancer lines. Enhanced activity was related to the presence of both cytokines on the same single-chain molecule and was not attributed to enhanced binding capacity. Killing was receptor specific. Cytotoxicity could be blocked with anti-EGF and anti - IL-13 antibodies. In vivo, DTEGF13, but not monospecific DTEGF or DTIL13, significantly inhibited the growth of established PC-3 tumors in nude mice (P < 0.0001). Conclusions: These data show for the first time that simultaneous targeting of cytokine receptors with two ligands on the same molecule has pronounced anticancer advantages. In an animal model in which human DTEGF13 is cross-reactive with mouse, DTEGF13 was highly effective in checking aggressive prostate tumor progression and was reasonably tolerated.