Chronic graft-versus-host-disease (cGVHD) can cause multiorgan system disease, typi-Key Points cally with autoimmune-like features, resulting in high mortality and morbidity caused • Low doses of donor iNKT by treatment limitations. Invariant natural killer T cells (iNKTs), a small population infusion prevent and reverse characterized by expression of a semi-invariant T-cell receptor, rapidly produce copious murine cGVHD. amounts of diverse cytokines on activation that exert potent immune regulatory function. • iNKT efficacy in treating Here, we show that iNKTs are significantly reduced in a cGVHD murine model that recapitulates several aspects of autoimmunity and organ fibrosis observed in patients established cGVHD is with cGVHD. Low iNKT infused doses effectively prevented and, importantly, reversed dependent on donor Treg established cGVHD, as did third-party iNKTs. iNKTs suppressed the autoimmune response expansion. by reducing the germinal center (GC) reaction, which was associated with an increase in total Tregs and follicular Tregs (Tfr) that control the GC reaction, along with pathogenic antibody production. Treg depletion during iNKT infusions completely abolished iNKT efficacy in treating cGVHD. iNKT cell interleukin 4 production and GC migration were critical to cGVHD reversal. In vivo stimulation of iNKT cells by a-galactosyl-ceramide was effective in both preventing and treating cGVHD. Together, this study demonstrates iNKT deficiency in cGVHD mice and highlights the key role of iNKTs in regulating cGVHD pathogenesis and as a potentially novel prophylactic and therapeutic option for patients with cGVHD.