The vectorial transport of ions and water across epithelial cells depends to a large extent on the co-ordination of the apical and basolateral ion fluxes with energy supply. In this work we provide the first evidence for a regulation by the 5'-AMP-activated protein kinase (AMPK) of the calcium activated potassium channel KCa3.1 expressed at the basolateral membrane of a large variety of epithelial cells. Inside-out patch clamp experiments performed on HEK cells stably transfected with KCa3.1 first revealed a decrease in KCa3.1 activity following the internal addition of AMP at a fixed ATP concentration. This effect was dose dependent with half inhibition at 140 µM AMP in 1 mM ATP. Evidence for an interaction between the C-terminal region of KCa3.1 and the 1 subunit of AMPK was next obtained by two-hybrid screening and pull-down experiments. Our two-hybrid analysis confirmed in addition that the amino acids extending from Asp³⁸⁰ to Ala⁴⁰⁰ in C-terminal were essential for the interaction AMPK-1/KCa3.1. Inside-out experiments on cells co-expressing KCa3.1 with the dominant negative AMPK-1-R299G mutant showed a reduced sensitivity of KCa3.1 to AMP, arguing for a functional link between KCa3.1 and the 1 subunit of AMPK. More importantly, co-immunoprecipitation experiments carried out on bronchial epithelial NuLi cells provided direct evidence for the formation of a KCa3.1/AMPK-1 complex at endogenous AMPK and KCa3.1 expression levels. Finally, treating NuLi monolayers with the membrane permeant AMPK activator AICAR caused a significant decrease of the KCa3.1 mediated short-circuit currents, an effect reversible by co-incubation with the AMPK inhibitor Compound-C. These observations argue for a regulation of KCa3.1 by AMPK in a functional epithelium through protein/protein interactions involving the 1 subunit of AMPK.