The vacuolar H⁺-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and non-volatile acid excretion. We previously showed that the A subunit in the cytoplasmic V₁ sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here we demonstrate that treatment of rabbit isolated perfused collecting ducts with the AMPK activator AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) inhibited V-ATPase-dependent H⁺ secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. As compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO₃^--containing media. Moreover, AICAR treatment of Clone C rabbit intercalated cells expressing WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in Clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues.