Granulocyte colony-stimulating factor (G-CSF) is a regulator of neutrophil production, function, and survival. Herein, we investigated the role of G-CSF in a murine model of human uveitis–experimental autoimmune uveoretinitis. Experimental autoimmune uveoretinitis was dramatically reduced in G-CSF–deficient mice and in anti–G-CSF monoclonal antibody–treated, wild-type (WT) mice. Flow cytometric analysis of the ocular infiltrate in WT mice with experimental autoimmune uveoretinitis showed a mixed population, comprising neutrophils, macrophages, and T cells. The eyes of G-CSF–deficient and anti–G-CSF monoclonal antibody–treated WT mice had minimal neutrophil infiltrate, but no change in other myeloid-derived inflammatory cells. Antigen-specific T-cell responses were maintained, but the differentiation of pathogenic type 17 helper T cells in experimental autoimmune uveoretinitis was reduced with G-CSF deficiency. We show that G-CSF controls the ocular neutrophil infiltrate by modulating the expression of C-X-C chemokine receptors 2 and 4 on peripheral blood neutrophils, as well as actin polymerization and migration. These data reveal an integral role for G-CSF–driven neutrophil responses in ocular autoimmunity, operating within and outside of the bone marrow, and also identify G-CSF as a potential therapeutic target in the treatment of human uveoretinitis.