Experimental autoimmune uveitis (EAU) is a well established model for immune-mediated organ-specific disease. Our group has recently shown that the M. leprae Hsp65 aggravated the uveitis in mice; in the present study, we evaluated the action of M. lepraeK⁴⁰⁹A mutant protein and the synthetic peptides Leader pep and K⁴⁰⁹A pep (covering amino acids residues 352–371 of WT and K⁴⁰⁹A proteins of M. leprae Hsp65, resp.) on the pathogenesis of EAU. Mice received the 161–180 IRBP peptide and B. pertussis toxin followed by the intraperitoneal inoculation of K⁴⁰⁹A protein or the Leader pep or K⁴⁰⁹A pep. The Leader pep aggravated the disease, but mice receiving the K⁴⁰⁹A pep did not develop the disease and presented an increase in IL-10 levels by spleen cells and a decrease in the percentage of CD4+ IFN-γ+ T cells. Moreover, animals receiving the Leader pep presented the highest scores of the disease associated with increase percentage of CD4+ IFN-γ+ T cells. These results would contribute to understanding of the pathogenesis of EAU and support the concept that immune responses to Hsp are of potential importance in exacerbating, perpetuating, or even controlling organ-restricted autoimmune diseases, and it is discussed the irreversibility of autoimmune syndromes.