Tumor protein D52 (D52) is constitutively expressed in healthy tissues and overexpressed in multiple cancers, including (but not limited to) breast, prostate and ovarian carcinomas. Although the normal functions of D52 are unknown, it is clear that increased D52 expression levels not only stimulate cell proliferation and metastasis, but also correlate with poor prognosis in a subset of breast cancer patients. The murine orthologs of D52 (mD52) shares 86% identity with its human counterpart (hD52) and mirrors hD52 expression patterns. The forced overexpression of mD52 induces anchorage-independent growth in vitro and promotes tumor formation as well as spontaneous metastasis in vivo. We have previously reported that the intramuscular administration of recombinant mD52 elicits immune responses capable of rejecting a challenge with tumor cells and preventing spontaneous metastasis only in 50% of mice. We hypothesized that mechanisms of peripheral tolerance dampen immune responses against mD52, thus limiting the protective effects of vaccination. To test this hypothesis, mice were depleted of CD25+ regulatory T cells (Tregs) and subcutaneously immunized with mD52 prior to a tumor challenge. The subcutaneous immunization failed to induce protective antitumor immunity unless accompanied by Treg depletion, which resulted in a rate of protection of 70% as compared with < 10% achieved in immunized Treg-repleted mice. The depletion of CD25+ Tregs did not inhibit the induction of immunological memory, since mice rejected a second tumor challenge administered weeks later. These data indicate that the induction of antitumor immune responses and immunologic memory by an mD52-based subcutaneous vaccination requires the depletion of CD25+ Tregs.