Sodium butyrate and its derivatives are useful therapeutic agents for the treatment of genetic diseases including urea cycle disorders, sickle cell disease, thalassemias, and possibly cystic fibrosis (CF). Butyrate partially restores cAMP-activated Cl⁻ secretion in CF epithelial cells by stimulating ΔF508 cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR) gene expression and increasing the amount of ΔF508-CFTR in the plasma membrane. Because the effect of butyrate on Cl⁻ secretion by renal epithelial cells has not been reported, we examined the effects of chronic butyrate treatment (15–18 h) on the function, expression, and localization of CFTR fused to the green fluorescent protein (GFP-CFTR) in stably transfected MDCK cells. We report that sodium butyrate reduced Cl⁻secretion across MDCK cells, yet increased apical membrane GFP-CFTR expression 25-fold and increased apical membrane Cl⁻ currents 30-fold. Although butyrate also increased Na-K-ATPase protein expression twofold, the drug reduced the activity of the Na-K-ATPase by 55%. Our findings suggest that butyrate inhibits cAMP-stimulated Cl⁻ secretion across MDCK cells in part by reducing the activity of the Na-K-ATPase.