Theophylline has been widely used in the treatment of airway disease but molecular mechanism has not been clearly elucidated. Previous studies have manifested that theophylline increases intracellular cAMP concentration. Because cystic fibrosis transmembrane conductance regulator(CFTR) is a cAMP-dependent Cl-channel that plays key roles in fluid secretion in vivo, we postulated that theophylline activates CFTR channel gating. We found (1) theophylline stimulated CFTR-mediated anion transport in a concentration-dependent manner, and CFTR specific blocker completely reversed the effect; (2) theophylline had no effect on △F508 or G551D mutant CFTR chloride channel activity; (3) theophylline had additive effect with forskolin(FSK) and 3-isobutyl-xanthin(IBMX), thus a di-rect binding activation mechanism was suggested. In conclusion, the results may provide a clue to elucidating the molecular mechanism of theophylline activities and theophylline may present a novel lead drug in treating CFTR-related disease. Theophylline has been widely used in the treatment of airway disease but the molecular mechanism has not been clearly elucidated. Previous studies have manifested that theophylline transients have increased in intracellular cAMP concentration. key roles in fluid secretion in vivo, we postulated that theophylline activates CFTR channel gating. We found (1) theophylline stimulated CFTR-mediated anion transport in a concentration-dependent manner, and CFTR specific blocker unbouled effect; (2) theophylline had no effect on Δ F508 or G551D mutant CFTR chloride channel activity; (3) theophylline had additive effect with forskolin (FSK) and 3-isobutyl-xanthin (IBMX), thus a di-rect binding activation mechanism was suggested. results may provide a clue to elucidating the molecular mechanism of theophylline activities and theophylline may present a novel lead drug in treating C FTR-related disease.