Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing adrenocorticotropin (ACTH) from the anterior pituitary gland and glucocorticoids from the adrenal cortex. Stress also activates the sympathetic nervous system, evoking adrenaline release from the adrenal medulla. Large conductance calcium- and voltage- activated potassium (BK) channels have been implicated in regulation of cellular excitability in these systems. Here, we examine the functional role of BK channels in HPA axis regulation in vivo using female mice genetically deficient (BK^-/-) for the pore-forming subunits of BK channels. BK^-/- phenotype in the HPA was confirmed by immunocytochemistry, Western blot analysis and corticotrope patch-clamp recording. Restraint stress-induced plasma concentrations of ACTH and corticosterone (CORT) were significantly blunted in BK^-/- mice compared to wild type (WT) controls. This stress hyporesponsiveness was associated with reduced activation of hypothalamic paraventricular nucleus (PVN) neurones. Basal expression of corticotropin releasing hormone (CRH), but not arginine vasopressin (AVP) mRNA in the PVN was significantly lower in BK^-/- mice compared to WT controls. Total anterior pituitary ACTH peptide content, but not POMC mRNA expression or corticotrope number, was significantly reduced in BK^-/- mice compared to WT. However, anterior pituitary corticotropes from BK^-/- mice fully supported ACTH output, releasing a significantly greater proportion of stored ACTH in response to secretagogue in vitro compared to WT. These results support an important role for BK channels in both the neural circuitry and endocrine output of the HPA axis, and indicate that the stress hyporesponsiveness in BK^-/- mice primarily results from reduced activation of hypothalamic PVN neurosecretory neurones.