Regulatory T cells (Treg) are supportive to cancer development in most tissues, but their role in colitis-associated colon cancer (CAC) remains unclear. In this study, we investigated the role of CD4⁺Foxp3⁺ Treg in a mouse model of CAC and in patients with colon cancer. These Treg were increased strongly in number in a mouse model of CAC and in the peripheral blood of patients with colon cancer, exhibiting an activated phenotype as defined by elevated expression of GARP, CD103, CTLA-4, and IL10, along with an increased suppressive effect on the proliferation and Th1 cytokine expression of CD4⁺CD25⁻ responder T cells ex vivo. Transient ablation of CD4⁺Foxp3⁺ Treg during tumor development in the CAC model suppressed tumor outgrowth and distribution, accompanied by an increased number of CD8⁺IFNγ/granzyme B-producing effector T cells. Conversely, inactivation of IL10 in Treg did not elevate the antitumor response but instead further boosted tumor development. Our results establish a tumor-promoting function for Treg during CAC formation, but they also suggest that a selective, transient ablation of Treg can evoke antitumor responses, with implications for immunotherapeutic interventions in patients with CAC. Cancer Res; 74(16); 4258–69. ©2014 AACR.