Abstract Description In autoimmune conditions, Th17 cells aberrantly accumulate and damage multiple types of organ. Diverse modalities targeting Th17 cells are under being tested for the therapeutics development, but only few options are available in clinic. Daphnetin, a major compound of Daphne jejudoensis, has been reported to alleviate Th17-mediated autoimmune diseases in mouse, but the precise mechanisms remains elusive. We aimed to evaluate the anti-Th17 effect of daphnetin and clarify the underlying mechanism. Our results showed that naïve CD4+ T cells were barely differentiated into Th17 cells in vitro upon daphnetin treatment. Consistently, daphnetin inhibited the transcription of BATF, IL23R, IRF4 and downregulated the protein levels of RORγt and phosphorylated STAT3 in CD4+ T cells. Metabolically, daphnetin undermined mitochondrial functional capacity through diminishing mitochondrial mass and membrane potential, presumably resulting from LXRβ activation. To validate the therapeutic effect in vivo, we treated an imiquimod (IMQ)-applied mice with daphnetin and found that clinical severity was dramatically alleviated compared to the untreated mice, with markedly reduced levels of IL-17A protein in tissue lysates. Collectively, these findings demonstrate that daphnetin may serve as a mitochondria inhibitor that suppresses the differentiation of Th17 cell. Further investigation will underscore the potential of daphnetin for the treatment of Th17-mediated autoimmune diseases. Funding Sources This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00334746) Topic Categories Therapeutic Approaches to Autoimmunity (THER)