Stimulation of several TNF receptor family proteins has been shown to dampen inflammatory disease in murine models through augmenting the number and/or activity of regulatory T cells (Tregs). We recently found that one molecule, 4-1BB, used binding to Galectin-9 to exert its immunosuppressive effects and drive expansion of CD8(+)Foxp3(-) Tregs. We now show that ligation of another TNFR family molecule, DR3, which has previously been found to strongly expand CD4(+)Foxp3(+) Tregs and suppress inflammation, also requires Galectin-9. We found that the extracellular region of DR3 directly binds to Galectin-9, and that Galectin-9 associates with DR3 in Tregs. From studies in vitro with Galectin-9(-/-) CD4(+) T cells and Tregs, we found that stimulatory activity induced by ligating DR3 was in part dependent on Galectin-9. In vivo, in a model of experimental autoimmune encephalomyelitis, we show that an agonist of DR3 suppressed disease, correlating with expansion of CD4(+)Foxp3(+) Tregs, and this protective effect was lost in Galectin-9(-/-) mice. Similar results were seen in an allergic lung inflammation model. Thus, we demonstrate a novel function of Galectin-9 in facilitating activity of DR3 related to Treg-mediated suppression.