Immune regulation has been shown to be involved in the progressive growth of some murine tumours. Interruption of immune regulatory pathways via CTL-associated antigen-4 (CTLA-4) blockade or removal of CD4(+) CD25(+) regulatory T (Treg) cells appears to be a promising strategy for cancer immunotherapy. In this study, we tested the hypothesis that the combination of CTLA-4 blockade and depletion of Treg cells would improve the potency of dendritic cell (DC)-based vaccine in a clinically relevant mouse model, which is transgenic for both carcinoembryonic antigen (CEA) and HLA-A2 for the treatment of colon carcinoma in a therapeutic setting. We found that administration of anti-CD25 antibody prior to vaccination or systemic administration of anti-CTLA-4 antibody with the vaccine improved tumour-free survival against CEA-expressing tumours compared with mice immunized with DC-based vaccine alone. However, the efficacy of the vaccine proved to be most effective when anti-CTLA-4 antibody was combined with Treg inhibition. This vaccination strategy dramatically improved the tumour-free survival and allowed the development of long-lasting immune responses. The combined vaccination strategy resulted in increased secretion of IFN-gamma and enhanced HLA-A2-restricted CEA-specific CTL responses. Furthermore, coadministration of anti-CD25 and anti-CTLA-4 antibodies along with the vaccine was effective against more advanced tumours. These results provide evidence that simultaneous blockade of T-cell regulatory pathways is a promising approach for the induction of therapeutic antitumour immunity against CEA(+) colon carcinoma.