T_regs play a crucial role in the maintenance of intestinal immune homeostasis. However, significant numbers of Foxp3⁺ T_regs accumulate in the inflamed lesions in experimental colitis and in IBD patients. T_reg production of the proinflammatory cytokines IFN-γ and/or IL-17 may arguably explain their ineffectiveness in suppressing intestinal inflammation. However, it remains unknown whether iT_reg and tT_reg produce proinflammatory cytokines and how TLR signaling regulates this process. Here, we found that Foxp3⁺T_regs were increased in the intestines of B6.TLR4^−/− and B6.IL-10^−/− mice when compared with WT B6 mice. TLR4^−/− and IL-10^−/− resulted in more T_regs within inflamed intestines. The majority of Foxp3⁺ T_regs in the spleen was Helios⁺Nrp1⁺, whereas most Foxp3⁺ T_regs in the intestinal LP were Helios⁻Nrp1⁻. More Helios⁺Nrp1⁺ T_regs expressed IFN-γ and/or IL-17 than did Helios⁻Nrp1⁻ T_regs in the spleen and intestine, which was increased with TLR4^−/−. TLR4 signaling in T cells and APCs inhibited Foxp3⁺ induction via MyD88-dependent, TRIF-independent pathways, which was negatively regulated by SOCS3. Collectively, these data demonstrate Helios⁺Nrp1⁺ tT_regs and Helios⁻Nrp1⁻ iT_regs produce proinflammatory cytokines in the intestines during inflammation, which was regulated by TLR4 signaling.