Though TGF-beta inhibition enhances antitumor immunity mediated by CD8(+) T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF-beta blockade, we tested the effect of anti-TGF-beta combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti-TGF-beta antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti-TGF-beta antibodies compared to vaccinated mice without anti-TGF-beta, suggesting that anti-TGF-beta synergistically enhanced irradiated tumor vaccine efficacy. CD8(+) T-cell depletion completely abrogated the vaccine efficacy, and so protection required CD8(+) T cells. Depletion of CD25(+) T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti-TGF-beta did not change the number of CD25(+) T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d-restricted NKT cells, which have been reported to induce TGF-beta production by MDSC through an IL-13-IL-4R-STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti-TGF-beta enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF-beta levels found in patients with cancer and that the effect is not dependent on TGF-beta solely from CD4(+)CD25(+) T regulatory cells or the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway.