We investigated the therapeutic effect of recombinant thymosin β4 (rTβ4) on rabbit autoimmune dacryoadenitis, an animal model of SS dry eye, and explore its mechanisms.Rabbits were treated topically with rTβ4 or PBS solution after disease onset for 28 days, and clinical scores were determined by assessing tear secretion, break-up time, fluorescein, hematoxylin and eosin staining, and periodic acid-Schiff. The expression of inflammatory mediators in the lacrimal glands were measured by real-time PCR. The expression of T helper 17 (Th17) cell-related transcription factors and cytokines were detected by real-time PCR and Western blotting. The molecular mechanism underlying the effects of rTβ4 on Th17 cell responses was investigated by Western blotting.Topical administration of rTβ4 after disease onset efficiently ameliorated the ocular surface inflammation and relieved the clinical symptoms. Further analysis revealed that rTβ4 treatment significantly inhibited the expression of Th17-related genes (RORC, IL-17A, IL-17F, IL-1R1, IL-23R, and granulocyte-macrophage colony-stimulating factor) and IL-17 protein in lacrimal glands, and meanwhile decreased the inflammatory mediators expression. Mechanistically, we demonstrated that rTβ4 repressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3) both in vivo and in vitro. Activation of the STAT3 signal pathway by Colivelin partly reversed the suppressive effects of rTβ4 on IL-17 expression in vitro.rTβ4 could alleviate ongoing autoimmune dacryoadenitis in rabbits, probably by suppressing Th17 response via partly affecting the STAT3 pathway. These data may provide a new insight into the therapeutic effect and mechanism of rTβ4 in dry eye associated with Sjögren's syndrome.