The development, progression and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous recurrent uveitis (ERU) to the retinal proteome. Cellular retinaldehyde binding protein (cRALBP) had not been previously described as autoantigen, but subsequent characterization in ERU horses revealed B- and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species, with typical tissue lesions at cRALBP-expression sites. The autoantibody profiling outlined here could be employed in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers.