Uveitis is the most common cause in inflammatory eye diseases that can lead to visual impairment even blindness worldwide. T helper (Th) 17 and regulatory T (Treg) cells are critical mediators for immune response. Notch signaling can regulate the cell differentiation, playing a role in the pathogenesis of the diseases. In this study, we measured the expression levels of Notch1, DLL4, IL-10, IL-17, RORγt and Foxp3 in T cells from lymph node, spleen and eye tissues in experimental autoimmune uveitis (EAU) rats in vitro, determined the ratios of CD4/CD8 and Th17/Treg. Moreover, we also investigated the effect of Notch signaling inhibitor N-(N-(3,5-Difluorophenacetyl-L-alanyl))-S-phenylglycine t-Butyl Ester (DAPT) on Notch1, DLL4 expression and on Th17, Treg cell differentiation. The results indicated that the pathogenesis of uveitis accompanied by the elevated expression of Notch1, DLL4, IL-10, IL-17, RORγt, and Foxp3 as well as the imbalanced CD4/CD8 and Th17/Treg ratios. By contrast, inhibition of Notch signaling by DAPT can efficiently decrease Th17 cell response, downregulate the expression of Notch1, DLL4, IL-17 and the transcription of RORγt, reduce Th17 levels and restore the CD4/CD8, Th17/Treg balance. Moreover, DAPT can also inhibit Th17 cell differentiation in healthy rats, though the inhibitory capacity of Th17, Treg differentiation is less than that in EAU rats. Overall, Notch signaling activation can lead to the disturbed Th17/Treg balance in uveitis, whereas inhibition of Notch signaling can ameliorate the inflammatory response and may be a potential immunoregulatory strategy in patients with uveitis.