The local interaction between retinal pigment epithelium (RPE) and immigrated effector T cells is crucial for the pathogenesis of autoimmune uveitis. After being activated by the pattern recognition receptors (PRRs) signaling pathway, RPE can present the antigen reactivated invading autoreactive T cells, resulting in uveitis. In the present study, we showed that the transfection of chitosan-loaded TLR3-siRNA toward RPE could effectively remit experimental autoimmune uveitis (EAU) in B10RIII mice. Initially, we verified the constitutive expression of Tlr3 in RPE at high levels, which was not altered in response to TNFα, IFNγ and IL-17A treatments. Compared with other TLRs, the activation of TLR3 signaling following polyIC treatment resulted in increased IL-6 and IFNγ secretion from and MHCII expression on RPE. It is polyIC-, but not other TLR ligands, treated RPE showed significant synergetic effect with IL-17 on stimulating RPE secreting CXCL8 and CCl2, which might be resulted from elevated Il17ra expression in RPE following polyIC treatment. Furthermore, polyIC-treated RPE caused a robust stimulation of differentiation of CD4 cell toward Th1 or Th17 cells, in addition to the secretion of the cytokines IFNγ and IL-17. The in vitro knockdown of TLR3 expression in RPE by chitosan/TLR3-siRNA transfection could effectively block polyIC-induced MHCII expression, pro-inflammatory cytokine secretion and autoreactive CD4 cell activation. Studies conducted in firefly luciferase gene transgenic mice demonstrated that the subretinal CS/Luci-siRNA transfection specifically reduced the luciferase activity in RPE but not in the liver and spleen. Finally, the CS/TLR3-siRNA was locally administered in the EAU induced B10RIII mice. The results revealed that chitosan-mediated TLR3-siRNA transfection had a significant therapeutic effect on either delaying the outbreak or remitting the severity of uveitis.