Immune checkpoint blockade using antibodies to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) benefits a limited number of cancer patients. SS1P and LMB-100 are immunotoxins that target mesothelin. We observed delayed responses to SS1P in patients with mesothelioma suggesting that anti-tumor immunity was induced. Our goal was to stimulate anti-tumor immunity by combining SS1P or LMB-100 with anti-CTLA-4. We constructed a BALB/c breast cancer cell line expressing human mesothelin (66C14-M) which was implanted in one or two locations. SS1P or LMB-100 was injected directly into established tumors and anti-CTLA-4 administered i.p. In mice with two tumors, one tumor was injected with immunotoxin and the other was not. The complete regression rate was 86% for the injected tumors and 53% for the uninjetced tumors. No complete regressions occurred when drugs were given separately. In regressing tumors, dying and dead tumor cells were intermingled with PMNs and surrounded by a collar of admixed eosinophils and mononuclear cells. Tumor regression was associated with increased numbers of tumor infiltrating CD8+ cells and blocked by administration of antibodies to CD8. Surviving mice were protected from tumor rechallenge by 66C14 cells not expressing mesothelin, indicating the development of anti-tumor immunity. The anti-tumor effect was abolished when a mutant non-cytotoxic variant was used instead of LMB-100, showing that the anti-tumor response is not mediated by recognition of a foreign bacterial protein. Our findings support developing a therapy composed of immunotoxins and checkpoint inhibitors for patients.