The anterior pituitary corticotroph is a major control point for the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the neuroendocrine response to stress. Although corticotrophs are known to be electrically excitable, ion channels controlling the electrical properties of corticotrophs are poorly understood. Here, we exploited a lentiviral transduction system to allow the unequivocal identification of live murine corticotrophs in culture. We demonstrate that corticotrophs display highly heterogeneous spontaneous action-potential firing patterns and their resting membrane potential is modulated by a background sodium conductance. Physiological concentrations of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) cause a depolarization of corticotrophs, leading to a sustained increase in action potential firing. A major component of the outward potassium conductance was mediated via intermediate conductance calcium-activated (SK4) potassium channels. Inhibition of SK4 channels with TRAM-34 resulted in an increase in corticotroph excitability and exaggerated CRH/AVP-stimulated ACTH secretion in vitro. In accordance with a physiological role for SK4 channels in vivo, restraint stress-induced plasma ACTH and corticosterone concentrations were significantly enhanced in gene-targeted mice lacking SK4 channels (Kcnn4(-/-)). In addition, Kcnn4(-/-) mutant mice displayed enhanced hypothalamic c-fos and nur77 mRNA expression following restraint, suggesting increased neuronal activation. Thus, stress hyperresponsiveness observed in Kcnn4(-/-) mice results from enhanced secretagogue-induced ACTH output from anterior pituitary corticotrophs and may also involve increased hypothalamic drive, thereby suggesting an important role for SK4 channels in HPA axis function.