AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilisation. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O₂ deficiency glycolytic degradation of glucose leads to formation of lactate and H⁺, thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H⁺ export. The present study explored whether AMPK influences Na⁺/H⁺ exchanger (NHE) activity and/or Na⁺-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH_i) was estimated utilizing BCECF fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺-dependent realkalinization (DpH_i) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH_i and DpH_i in those cells were significantly increased by treatment with AMPK stimulator AICAR (1 mM) and significantly deceased by AMPK inhibitor compound C (10 mM). The effect of AICAR on pH_i and DpH_i was blunted in the presence of the Na⁺/H⁺ exchanger inhibitor cariporide (10 mM), but not by the H⁺ ATPase inhibitor bafilomycin (10 nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.