The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or GPI-anchors. Overexpressed in ovarian tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated anti-tumor therapies. We sought to engineer anthrax toxin protective antigen (PrAg), which is typically proteolytically activated on the cell surface by the proprotein convertase furin, to instead be activated by tumor cell-expressed membrane-anchored serine proteases testisin, hepsin, and matriptase to function as a tumoricidal agent. PrAg's native activation sequence was mutated to sequences reported to be cleaved by testisin, hepsin, and matriptase to generate novel PrAg proteins. The novel PrAg proteins were resistant to furin cleavage in vitro, yet able to be cleaved by testisin, hepsin, and matriptase in vitro and in cell culture to mediate increased killing of tumor cells. When combined with FP59, a chimeric lethal factor-Pseudomonas exotoxin fusion protein, the mutant PrAg toxins (PrAg proteins and FP59) were cytotoxic to multiple human tumor cell lines, including a panel of human ovarian tumor cells. Treatment with the novel PrAg toxins potently attenuated subcutaneous xenograft tumor growth, as well as intraperitoneal tumor growth in an orthotopic xenograft model of ovarian cancer. Moreover, treatment with one of the novel PrAg toxins effectively reduced established intraperitoneal ovarian tumor burden in the orthotopic xenograft model. The results from this study suggest that anthrax toxin PrAg can be engineered to target and become activated by tumor cell-expressed membrane-anchored serine proteases, and may potentially function as potent, protease-activated, tumor-targeted therapeutic(s) for the treatment of ovarian cancer.