Recent studies found that palmitic acid (PA), the most abundant fatty acid in human body, was increased in uveitis patients. However, its exact effect on uveitis has not been clarified. In this study, experimental autoimmune uveitis (EAU), an animal model of human uveitis, was successfully induced with interphotoreceptor retinoid-binding protein (IRBP) 651-670 and pertussis toxin. The immunized mice were treated with daily intragastric PA or vehicle from day 1-14. The results showed that PA could aggravate EAU activities and increase the proportion of T helper (Th) 17 cells as well as mRNA expression level of Il17a. There were no significant changes in Th1/Treg cell responses between these two groups. In vitro experiments showed that PA treatment could promote IRBP-specific Th17 cell response in association with increased proportion of Th17 cells as well as up-regulated expression of IL-17A. Proteomics showed an increased expression of stimulator of interferon genes protein (STING) in PA-treated mice as compared to vehicle-treated mice. H-151, a potent antagonist of STING, attenuated the activities of EAU and Th17 cell responses induced by PA. Moreover, NF-κB/IL-6 signaling pathway was found to be downregulated after H-151 treatment. Collectively, PA could exacerbate EAU severity possibly through the activation of Th17 cells mediated by up-regulating STING.