Context: Inactivating germline mutations of the probable tumor suppressor gene Armadillo Repeat Containing 5 (ARMC5) have recently been identified as a genetic cause of macronodular adrenal hyperplasia (MAH). Objective: We searched for ARMC5 mutations in a large cohort of patients with MAH. The clinical phenotype of patients with and without ARMC5 mutations was compared. Methods: Blood DNA from 34 MAH patients was genotyped using Sanger sequencing. Diurnal serum cortisol measurements, plasma ACTH levels, urinary steroids, 6-day Liddle's test, adrenal computed tomography, and weight of adrenal glands at adrenalectomy were assessed. Results: Germline ARMC5 mutations were found in 15 out of 34 patients (44.1%). In silico analysis of the mutations indicated that 7 (20.6%) predicted major implications for gene function. Late-night cortisol levels were higher in patients with ARMC5 damaging mutations compared to those without and/or with non-pathogenic mutations (14.5±5.6 vs. 6.7±4.3, p<0.001). All patients carrying a pathogenic ARMC5 mutation had clinical Cushing's syndrome (7/7, 100%) compared to 14/27 (52%) of those without or with mutations that were predicted to be benign (p=0.029). Repeated measures analysis showed overall higher urinary 17-hydroxycorticosteroids and serum cortisol values in the patients with ARMC5 damaging mutations during the 6 day Liddle's test (p=0.0002). Conclusions: ARMC5 mutations are implicated in clinically severe Cushing's syndrome associated with MAH. Knowledge of a patient's ARMC5 status has important clinical implications for the diagnosis of Cushing's syndrome and genetic counseling of patients and their families.