Sodium 4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to treat patients with urea cycle enzyme deficiencies and is being evaluated in the management of sickle cell disease, thalassemia, cancer, and cystic fibrosis (CF). Because relatively little is known about the effects of PBA on the expression and function of the wild-type CF transmembrane conductance regulator (wt CFTR), the goal of this study was to examine the effects of PBA and related compounds on wt CFTR-mediated Cl⁻secretion. To this end, we studied Calu-3 cells, a human airway cell line that expresses endogenous wt CFTR and has a serous cell phenotype. We report that chronic treatment of Calu-3 cells with a high concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but not of sodium acetate reduced basal and 8-(4-chlorophenylthio)-cAMP-stimulated Cl⁻ secretion. Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and increased the intensity of CFTR staining in the apical plasma membrane. PBA also increased protein expression of Na⁺-K⁺-ATPase. PBA reduced CFTR Cl⁻currents across the apical membrane but had no effect on Na⁺-K⁺-ATPase activity in the basolateral membrane. Thus a high concentration of PBA (5 mM) reduces Cl⁻ secretion by inhibiting CFTR Cl⁻currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05–2 mM) had no effect on cAMP-stimulated Cl⁻ secretion across Calu-3 cells. We conclude that PBA concentrations in the therapeutic range are unlikely to have a negative effect on Cl⁻ secretion. However, concentrations >5 mM might reduce transepithelial Cl⁻ secretion by serous cells in submucosal glands in individuals expressing wt CFTR.