Background <p>Uveitis is a common recurrent autoimmune disease that seriously endangers the visual health of patients. MicroRNAs (miRNAs) are closely related to a series of autoimmune diseases.</p> Methods <p>The present study aimed to investigate the effect of miR-30b-5p on experimental autoimmune uveitis (EAU) and its role in Notch signal activation as well as T helper (Th) cell differentiation, the relationship between miR-30b-5p levels and Notch signal activation, as well as Th cell differentiation in uveitis was further explored through flow cytometry, Immunohistochemistry immunofluorescence staining, PCR Array, and Ingenuity Pathway Analysis, and other technical methods to determine the fidelity of miR-30b-5p strategies in treating uveitis in vivo and in vitro<i>.</i></p> Results <p>We demonstrated that ocular inflammation was significantly alleviated in EAU rats after miR-30b-5p intervention. miR-30b-5p could effectively inhibit Notch signaling activation and Th17 cell differentiation both in vitro and in vivo, and the Th17/Treg ratios were also notably decreased. Moreover, both Notch signaling and Th17 activation pathways were enriched and activated, in which Notch1 was the upstream regulatory molecule of Dll4 and IL-10 was an up-regulated upstream regulatory network molecule. Furthermore, miR-30b-5p could significantly reduce apoptosis in vitro, and clinical in vitro cell studies have shown that inactivating Notch pathway can improve the imbalance of Th17/Treg and cell apoptosis in T lymphocytes of patients with uveitis.</p> Conclusions <p>Together these studies identify that miR-30b-5p can significantly inhibit Notch signaling activation and Th17 cell differentiation, thereby restoring the Th17/Treg balance to treat uveitis, which may provide new insights into treating uveitis using miRNA interfering strategies.</p> Graphical Abstract <p></p>