Autoantibodies to surface molecules on lymphocytes have already been described in various immune conditions, such as, autoimmune diseases, infections, and blood transfusions. Because T-cell costimulatory molecules play a central role in the immune response of T-cells, we investigated the presence of autoantibodies against T-cell costimulatory molecules in canine autoimmune diseases. In this study, we prepared recombinant proteins of CTLA-4 (CD152) and CD28 and investigated the presence of autoantibodies against them in serum samples obtained from dogs with various autoimmune diseases and from healthy dogs as controls, using the recombinant GST fusion proteins by ELISA. Anti-CTLA-4 antibodies were found in 31.8% of patients with rheumatoid arthritis, 20% of patients with systemic lupus erythematosus, 12.5% of patients with pemphigus, 0% of patients with immune-mediated hemolytic anemia, and 0% of healthy donors. Anti-CD28 antibodies were not found in any of the patients or healthy donors. The ELISA results were further confirmed by immunoblotting. The presence of anti CTLA-4 antibodies suggests the existence of a CTLA-4-specific immune response. The autoantibodies against CTLA-4, demonstrated here for the first time in canine autoimmune diseases, may modulate the immune response in dogs with autoimmune diseases.