Cryoablation of a solitary tumor mass releases intact tumor antigens and can induce protective antitumor immunity but has limited efficacy in the treatment of established metastatic cancer. Cyclophosphamide (Cy), an anticancer drug, selectively depletes regulatory T cells (T(reg)s) and attenuates suppression of antitumor immunity. We used a BALB/c mouse model of metastatic colon cancer to investigate the systemic antitumor effects of in situ cryotherapy alone or in combination with 200 mg/kg i.p. Cy. When combined with Cy, cryoablation was significantly more effective than either surgical excision or cautery at inducing systemic antitumor immunity, resulting in the cure of a fraction of animals with established metastatic disease and resistance to tumor rechallenge. Lymphocytes from cured animals contained an expanded population of tumor-specific, interferon-gamma producing T cells and transferred antitumor immunity to naive recipients. Depletion of CD8(+) cells significantly impaired the adoptive transfer of antitumor immunity. Furthermore, treatment with Cy and cryoablation was associated with a significant decrease in the ratio of regulatory to effector CD4(+) T cells. The combination of tumor cryoablation and Cy induces potent, systemic antitumor immunity in animals with established metastatic disease.