The Ca2+-activated potassium channel, KCa3.1, and the Ca2+ release-activated calcium channel, Orai, are implicated in the process of exocytic release of intracellular granules from mast cells. MUC5AC mucin is upregulated in asthmatic human airways, and is released from secretory human bronchial epithelial cells (HBECs) via exocytosis of intracellular granules. The central hypothesis tested here was that KCa3.1 and Orai channels modulate MUC5AC production and secretion from HBECs. Ion channel expression was examined in primary HBECs isolated from asthmatic and healthy donors and the human airway epithelial cell line, H292, by qPCR, western blotting and patch clamp electrophysiology. MUC5AC and KCa3.1 expression in bronchial biopsies were analysed using immunohistochemistry. Mucin production and secretion from in vitro cultures were quantified by lectin assay and immunohistochemistry. This study provides the first report of KCa3.1 expression and activity in primary HBECs. KCa3.1 mRNA was expressed to a similar extent in both asthmatic and healthy HBECs, and protein expression was evident in western blots. Asthmatic HBECs displayed significantly larger TRAM-34-sensitive KCa3.1 currents than healthy HBECs (P = 0.023). MUC5AC and KCa3.1 co-localised in approximately 50% of HBECs within bronchial biopsy samples in asthma and in health. Severe asthmatic bronchial epithelium displayed significantly higher levels of KCa3.1 (P = 0.001) and MUC5AC (P = 0.018) immunostaining in comparison to healthy epithelium. KCa3.1 and MUC5AC immunostaining in the bronchial epithelium correlated across the severities of asthma (P = 0.0211). KCa3.1 blockade did not inhibit amphiregulin-induced MUC5AC production and secretion from H292 cells, or IL-13-induced MUC5AC mRNA expression in primary HBECs. Despite detecting Orai protein and mRNA expression in primary HBECs, Orai currents could not be recorded. These data suggest that KCa3.1 expression in the bronchial epithelium in asthma has functional relevance, but is unlikely to regulate mucus production. The role of Orai channels in primary HBECs requires further investigation.