AMP-activated protein kinase (AMPK) is activated in response to fluctuations in cellular energy status caused by oxidative stress. One of its targets is the cystic fibrosis transmembrane conductance regulator (CFTR), which is the predominant Cl- secretory channel in colonic tissue. The aim of this study was to determine the role of AMPK in the modulation of colonic chloride secretion under conditions of oxidative stress and chronic inflammation. Chloride secretion and AMPK activity were examined in colonic tissue from adult IL-10-deficient and wild-type 129 Sv/Ev mice in the presence and absence of pharmacological AMPK inhibitors and activators, respectively. Apical levels of CFTR were measured in brush-border membrane vesicles. Cell culture studies in human colonic T84 monolayers examined the effect of hydrogen peroxide and pharmacological activation of AMPK on forskolin-stimulated chloride secretion. Inflamed colons from IL-10-deficient mice exhibited hyporesponsiveness to forskolin stimulation in association with reductions in surface CFTR expression and increased AMPK activity. Inhibition of AMPK restored tissue responsiveness to forskolin, whereas stimulation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) induced tissue hyporesponsivness in wild-type mice. T84 cells exposed to hydrogen peroxide demonstrated a time-dependent increase in AMPK activity and reduction of forskolin-stimulated chloride secretion. Inhibition of AMPK prevented the reduction in chloride secretion. Treatment of cells with the AMPK activator, AICAR, resulted in a decreased chloride secretion. In conclusion, AMPK activation is linked with reductions in cAMP-mediated epithelial chloride flux and may be a contributing factor to the hyporesponsiveness seen under conditions of chronic inflammation.