© Society for Leukocyte Biology. Tregs play a crucial role in the maintenance of intestinal immune homeostasis. However, significant numbers of Foxp3+Tregs accumulate in the inflamed lesions in experimental colitis and in IBD patients. Tregproduction of the proinflammatory cytokines IFN-γ and/or IL-17 may arguably explain their ineffectiveness in suppressing intestinal inflammation. However, it remains unknown whether iTregand tTregproduce proinflammatory cytokines and how TLR signaling regulates this process. Here, we found that Foxp3+Tregswere 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 Tregswithin inflamed intestines. The majority of Foxp3+Tregsin the spleen was Helios-Nrp1-, whereas most Foxp3+Tregsin the intestinal LP were Helios+Nrp1+. More Helios-Nrp1-Tregsexpressed IFN-γand/or IL-17 than did Helios+Nrp1+Tregs 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+tTregsand Helios--Nrp1-iTregsproduce proinflammatory cytokines in the intestines during inflammation, which was regulated by TLR4 signaling.