The adrenal glands are essential endocrine organs that secrete key hormones maintaining physiological homeostasis. Herein, we established expandable three-dimensional (3D) human adrenocortical organoid (ACO) cultures that preserved the characteristic of zona fasciculata cell lineages and retained their capacity to produce cortisol. The ACOs could secrete glucocorticoids in response to physiological stimuli and thus rescue adrenalectomized mice, indicating their potential for the treatment of primary adrenal insufficiency. Furthermore, by introducing a hotspot pathogenic variant (PRKACA L206R) identified in Cushing’s syndrome, we achieved the organoid disease modeling of cortisol-producing adenomas. In summary, this study establishes a human organoid platform to explore homeostasis and dysfunction in adrenal glands, suggesting future applications in disease modeling and regenerative medicine.