BackgroundThe activation of CD8+T cells and their trafficking to the skin through JAK-STAT signaling play a central role in the development of vitiligo. Thus, targeting this key disease pathway with innovative drugs is an effective strategy for treating vitiligo. Natural products isolated from medicinal herbs are a useful source of novel therapeutics. Demethylzeylasteral (T-96), extracted from Tripterygium wilfordii Hook F, possesses immunosuppressive and anti-inflammatory properties.MethodsThe efficacy of T-96 was tested in our mouse model of vitiligo, and the numbers of CD8+T cells infiltration and melanocytes remaining in the epidermis were quantified using whole-mount tail staining. Immune regulation of T-96 in CD8+T cells was evaluated using flow cytometry. Pull-down assay, mass spectrum analysis, molecular docking, knockdown and overexpression approaches were utilized to identify the target proteins of T-96 in CD8+T cells and keratinocytes.ResultsHere, we found that T-96 reduced CD8+T cell infiltration in the epidermis using whole-mount tail staining and alleviated the extent of depigmentation to a comparable degree of tofacitinib (Tofa) in our vitiligo mouse model. In vitro, T-96 decreased the proliferation, CD69 membrane expression, and IFN-γ, granzyme B, (GzmB), and perforin (PRF) levels in CD8+T cells isolated from patients with vitiligo. Pull-down assays combined with mass spectrum analysis and molecular docking showed that T-96 interacted with JAK3 in CD8+T cell lysates. Furthermore, T-96 reduced JAK3 and STAT5 phosphorylation following IL-2 treatment. T-96 could not further reduce IFN-γ, GzmB and PRF expression following JAK3 knockdown or inhibit increased immune effectors expression upon JAK3 overexpression. Additionally, T-96 interacted with JAK2 in IFN-γ-stimulated keratinocytes, inhibiting the activation of JAK2, decreasing the total and phosphorylated protein levels of STAT1, and reducing the production and secretion of CXCL9 and CXCL10. T-96 did not significantly inhibit STAT1 and CXCL9/10 expression following JAK2 knockdown, nor did it suppress upregulated STAT1-CXCL9/10 signaling upon JAK2 overexpression. Finally, T-96 reduced the membrane expression of CXCR3, and the culture supernatants pretreated with T-96 under IFN-γ stressed keratinocytes markedly blocked the migration of CXCR3+CD8+T cells, similarly to Tofa in vitro.ConclusionOur findings demonstrated that T-96 might have positive therapeutic responses to vitiligo by pharmacologically inhibiting the effector functions and skin trafficking of CD8+T cells through JAK-STAT signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-023-04293-2.