PURPOSE: Immunoregulatory and suppressive mechanisms represent major obstacles to the success of immunotherapy in cancer patients. We have shown that the combination of radiotherapy to the primary tumor and CTL-associated protein 4 (CTLA-4) blockade induces antitumor immunity, inhibiting metastases and extending the survival of mice bearing the poorly immunogenic and highly metastatic 4T1 mammary carcinoma. Similarly to patients with metastatic cancer, however, mice were seldom cured. Here we tested the hypothesis that invariant natural killer T (iNKT) cells, a subset with unique regulatory functions, can regulate the response to radiotherapy and CTLA-4 blockade. EXPERIMENTAL DESIGN: The growth of 4T1 primary tumors and lung metastases was compared in wild-type and iNKT cell-deficient (iNKT-/-) mice. Treatment was started on day 13 when the primary tumors were palpable. Mice received radiotherapy to the primary tumor in two doses of 12 Gy in combination or not with 9H10 monoclonal antibody against CTLA-4. Response to treatment was assessed by measuring primary tumor growth delay/regression, survival, and number of lung metastases. RESULTS: The response to radiotherapy plus 9H10 was markedly enhanced in the absence of iNKT cells, with 50% of iNKT-/- versus 0% of wild-type mice showing complete tumor regression, long-term survival, and resistance to a challenge with 4T1 cells. Administration of the iNKT cell activator alpha-galactosylceramide did not enhance the response of wild-type mice to radiotherapy plus 9H10. Tumor-infiltrating iNKT cells were markedly reduced in wild-type mice treated with radiotherapy plus 9H10. CONCLUSIONS: iNKT cells play a major role in regulating the response to treatment with local radiotherapy and CTLA-4 blockade.