—Although immunoapheresis removing autoantibodies against the second extracellular domain of β₁-adrenergic receptors (ARs) improves cardiac function in patients with cardiomyopathy, the underlying mechanisms have not been defined. We examined the role of autoimmunity against the domain in the development of cardiac dysfunction in vivo. Japanese white rabbits were immunized with a synthetic peptide corresponding to the second extracellular loop of β₁-AR once a month with (β+biso rabbits, n=10) or without (β rabbits, n=13) bisoprolol treatment (2 mg/kg per day). Control rabbits received vehicle without bisoprolol treatment (n=13). Autoantibodies of IgG isotype against the domain were persistently detected in β and β+biso rabbits. Purified IgG from sera of β and β+biso rabbits increased cAMP production in a rabbit cardiac membrane preparation, which was blocked by bisoprolol. At 3 months, β-AR uncoupling with increased G protein–coupled receptor kinase 5 (GRK5) expression was found in β rabbits. At 6 months, left ventricular hypertrophy was noted with hemodynamic derangements in β rabbits. This was accompanied by decreased β₁-AR density and increased inhibitory G protein and GRK5 expression, which were related to marked decrease in membrane cAMP production. These changes in β rabbits at 6 months were prevented in β+biso rabbits. There was no difference in the plasma norepinephrine concentration in the 3 groups over the observation period. Thus, autoimmunity against the second extracellular loop of β₁-ARs induced profound β-AR desensitization and myocardial hypertrophy in vivo, associated with cardiac dysfunction. Sustained sympathomimetic-like actions of autoantibodies against the domain may be partly responsible for these changes.