Reduced signal at intercalated discs (IDs) and nuclear accumulation of the desmosomal protein plakoglobin (γ-catenin) in arrhythmogenic cardiomyopathy (ACM) have been linked to suppression of canonical Wnt/β-catenin signaling. In a chemical screen in a zebrafish model of ACM, we found that SB216763, annotated as an inhibitor of glycogen synthase kinase-3β (GSK-3β), prevents heart failure and reduces mortality. It also prevents arrhythmias and myocyte injury and apoptosis in cultured neonatal rat ventricular myocytes (NRVM) and intact mouse hearts expressing ACM mutations in 3 different desmosomal genes. Given the role of GSK-3β in the canonical Wnt/β-catenin pathway, we further examined GSK-3β in ACM pathogenesis. To determine if GSK-3β is redistributed in ACM, we immunostained myocardium from 11 patients who met diagnostic criteria, including 8 with desmosomal gene mutations. GSK-3β immunoreactive signal accumulated at ventricular myocyte IDs in the hearts of all patients with ACM but not in control hearts or samples from patients with end-stage ischemic, dilated or hypertrophic cardiomyopathies (n=5 for each) or cardiac sarcoidosis (n=10). The GSK-3β binding partner adenomatous polyposis coli (APC) also localized to cell-cell junctions in ACM hearts only, consistent with the presence of the GSK-3β/axin/APC complex at IDs in ACM. GSK-3β and APC also showed marked junctional localization in NRVM expressing mutant forms of plakoglobin (2057del2) or plakophilin2 (1851del123), and in the hearts of adult mice expressing 2057del2 plakoglobin and a cardiac myocyte-specific Dsg2 knock-in mouse, but not in normal controls cells or tissues. GSK-3β and APC distributions were normalized by SB216763 in these in vitro and in vivo models of ACM. Immunoblotting showed no apparent change in the total cellular content of GSK-3β in the presence of ACM-causing desmosomal gene mutations. However, in addition to restoring the normal cytoplasmic localization of the GSK-3β/APC complex, SB216763 also decreased the total cellular content of the active (non-phospho) form of GSK-3β in all 4 experimental models. These observations suggest a role for GSK-3β distribution in the ACM disease mechanism independent of the specific underlying desmosomal mutation.