Background/Aims: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (I_SC) in a human bronchial epithelial cell line (16HBE14o-), and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. Methods: The I_SC measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca²⁺]_i) and cAMP were also quantified. Results: Nobiletin stimulated a concentration-dependent increase in I_SC, which was due to Cl^- secretion. The increase in I_SC was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTR_inh-172), but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS), Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated I_SC was also sensitive to a protein kinase A (PKA) inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in I_SC in a cystic fibrosis (CF) cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca²⁺]_i. Conclusion: Nobiletin stimulated transepithelial Cl^- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl^- channels.