Abstract Ovarian cancer remains the most lethal gynecologic malignancy in the United States. Although many patients with advanced-stage disease initially respond to standard combinations of surgical and cytotoxic therapy, nearly 90% develop recurrence and inevitably die from the development of chemotherapy-resistant disease. The discovery of novel and effective therapy against chemotherapy-resistant/recurrent ovarian cancer remains a high priority. Using expression profiling, we and others have recently found claudin-3 and claudin-4 genes to be highly expressed in ovarian cancer. Because these tight junction proteins have been described as the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE), in this study we investigated the level of expression of claudin-3 and/or claudin-4 in chemotherapy-naïve and chemotherapy-resistant primary human ovarian cancers as well as their sensitivity to CPE treatment in vitro. We report that 100% (17 of 17) of the primary ovarian tumors tested overexpress one or both CPE receptors by quantitative reverse transcription-PCR. All ovarian tumors showed a dose-dependent cytotoxic effect to CPE in vitro. Importantly, chemotherapy-resistant/recurrent ovarian tumors were found to express claudin-3 and claudin-4 genes at significantly higher levels when compared with chemotherapy-naïve ovarian cancers. All primary ovarian tumors tested, regardless of their resistance to chemotherapeutic agents, died within 24 hours to the exposure to 3.3 μg/mL CPE in vitro. In addition, we have studied the in vivo efficacy of i.p. CPE therapy in SCID mouse xenografts in a highly relevant clinical model of chemotherapy-resistant freshly explanted human ovarian cancer (i.e., OVA-1). Multiple i.p. administration of sublethal doses of CPE every 3 days significantly inhibited tumor growth in 100% of mice harboring 1 week established OVA-1. Repeated i.p. doses of CPE also had a significant inhibitory effect on tumor progression with extended survival of animals harboring large ovarian tumor burdens (i.e., 4-week established OVA-1). Our findings suggest that CPE may have potential as a novel treatment for chemotherapy-resistant/recurrent ovarian cancer.