PURPOSE: The purpose of this study was to explore the underlying mechanism that Th17-like T follicular helper cells (Tfh) orchestrated by STING signaling have a pathogenic role in experimental autoimmune uveitis (EAU). METHODS: The differences of transcriptome and gene ontology (GO) pathway of Tfh between EAU and control mice were analyzed by single-cell RNA sequence (scRNA-seq) and bulk RNA sequence. Additionally, draining lymph nodes (DLNs) were extracted to verify the expression of IL-17A and IFN-γ in Tfh from EAU and control mice by flow cytometry. Then, the scRNA-seq and flow cytometry were used to explore the different proportion of Tfh between STING deficiency (Sting-/-) mice and wild type (WT) mice. In vitro, naïve CD4+ T cells were isolated from Sting-/- mice and WT mice to induce the Tfh under the induction condition. In addition, flow cytometry was used to detect the different induction ratio and the IL-17A expression between 2 groups of naïve CD4+ T cells. RESULTS: Compared with control mice, marked increase of Tfh was observed in EAU, accompanied by elevated levels of Th1 and Th17 cells. Moreover, Th17-related genes, such as Rorc, Il22, Il23r, Il17a, and Il17f, and the corresponding GO pathways were upregulated in Tfh from EAU. The scRNA-seq showed that a higher proportion of Tfh was observed in the DLNs from Sting-/- mice than WT mice, which was verified by flow cytometry. When STING was knocked out, the Tfh was characterized with upregulated Th17-related phenotype in vivo, and there was a higher induction ratio of Tfh whose IL-17A expression was significantly increased in vitro. Notably, the STING expression of CD4+ T cells was downregulated in the EAU. STING-deficient EAU mice displayed more severe retinal inflammation, characterized by massive infiltration of CD4+ T cells, including Th1 and Th17 subsets. Importantly, treatment with a STING agonist alleviated inflammation of EAU. CONCLUSIONS: Th17-like Tfh cells play a pathogenic role in the EAU. STING deficiency promotes the differentiation and phenotypic transformation of Th17-like Tfh cells, exacerbating the inflammatory response in EAU. These findings highlight the potential of targeting STING to modulate Tfh cells as a therapeutic strategy for uveitis.