The clinical importance of autoantibodies against the ganglionic acetylcholine receptor (gAChR) remains to be fully elucidated. We aimed to identify the clinical characteristics of autoimmune autonomic ganglionopathy (AAG) in patients with gAChR autoantibodies. For this cohort investigation, serum samples were obtained from patients with AAG between 2012 and 2018 in Japan. We measured the levels of autoantibodies against gAChRα3 and gAChRβ4 and evaluated clinical features, as well as assessing the laboratory investigation results among the included patients. A total of 179 patients tested positive for antibodies, including 116 gAChRα3-positive, 13 gAChRβ4-positive, and 50 double antibody-positive patients. Seropositive AAG patients exhibited widespread autonomic dysfunction. Extra-autonomic manifestations including sensory disturbance, central nervous system involvement, endocrine disorders, autoimmune diseases, and tumours were present in 118 patients (83%). We observed significant differences in the frequencies of several autonomic and extra-autonomic symptoms among the three groups. Our I-metaiodobenzylguanidine myocardial scintigraphy analysis of the entire cohort revealed that the heart-to-mediastinum ratio had decreased by 80%. The present study is the first to demonstrate that patients with AAG who are seropositive for anti-gAChRβ4 autoantibodies exhibit unique autonomic and extra-autonomic signs. Decreased cardiac uptake occurred in most cases, indicating that I- metaiodobenzylguanidine myocardial scintigraphy may be useful for monitoring AAG. Therefore, our findings indicate that gAChRα3 and gAChRβ4 autoantibodies cause functional changes in postganglionic fibres in the autonomic nervous system and extra-autonomic manifestations in seropositive patients with AAG.