The present study concerns the involvement of the ceramide produced through sphingomyelinase (SMase)-mediated catalysis in airway anion secretion of Calu-3 cells. Short-circuit current (I_sc) measurement revealed that isoproterenol (ISO, 0.1μM)-induced anion secretion was prevented by pretreatment with SMase (0.3U/ml, for 30min) from the basolateral but not the apical side, although basal and 1-ethyl-2-benzimidazolinone (1-EBIO, a Ca²⁺-activated K⁺ channel opener)-induced I_sc were unaffected. The effects of SMase were reproduced in responses to forskolin (20μM) or 8-bromo-cAMP (2mM). C₂-ceramide, a cell-permeable analog, also repressed the 8-bromo-cAMP-induced responses. Nystatin permeabilization studies confirmed that the SMase- and C₂-ceramide-induced repressions were due to hindrance of augmentation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance across the apical membrane. Further, SMase failed to influence K⁺ conductance across the basolateral membrane. These results suggest that the ceramide originating from basolateral sphingomyelin acts on activated CFTR from the cytosolic side, hindering anion secretion.