CD4Foxp3 regulatory T cells (T) are pivotal for the inhibition of autoimmune inflammatory responses. One way to therapeutically harness the immunosuppressive actions of T is to stimulate the proliferative expansion of TNFR2-expressing CD4Foxp3 T via transmembrane TNF (tmTNF). Here, we report that two-pore channel (TPC) inhibitors markedly enhance tmTNF expression on antigen-presenting cells. Furthermore, injection of TPC inhibitors including tetrandrine, or TPC-specific siRNAs in mice, increases the number of T in a tmTNF/TNFR2-dependent manner. In a mouse colitis model, inhibition of TPCs by tetrandrine markedly attenuates colon inflammation by expansion of T. Mechanistically, we show that TPC inhibitors enhance tmTNF levels by disrupting surface expression of TNF-α-converting enzyme by regulating vesicle trafficking. These results suggest that the therapeutic potential of TPC inhibitors is mediated by expansion of TNFR2-expressing T and elucidate the basis of clinical use in the treatment of autoimmune and other inflammatory diseases.