This study purposed to depict the transcriptional changes associated with autoimmune uveitis (AU) pathogenesis and identify potential therapeutic targets of this disease. An experimental AU (EAU) model was established with retina antigen and adjuvants. An EAU control group was established with adjuvant only to eliminate nonspecific effects. We conducted single-cell RNA sequencing (scRNA-seq) on cervical draining lymph node cells of EAU, EAU control, and normal mice to identify the EAU-associated transcriptional changes and the potential pathogenic molecules. Subsequent flow cytometry, adoptive transfer experiment, scRNA-seq analysis of human uveitis, and proliferation assessment were conducted to verify the function of the interested molecule in uveitis. The scRNA-seq data suggested that hypoxia-inducible factor 1 alpha (Hif1α) may participate in EAU pathogenesis via regulating T helper (Th)-17, Th1, and regulatory T cells. Hif1α inhibition alleviated EAU symptoms and regulated Th17, Th1, and regulatory T cell proportions. CD4+ T cells with repressed Hif1α expression failed to transfer EAU to naïve mice. In Vogt-Koyanagi-Harada disease, which is a human uveitis, Hif1α was also increased in CD4+ T cells and regulated their proliferation. The results indicate that Hif1α may participate in AU pathogenesis and are, thus, a potential therapeutic target.