Acidic luminal pH and low [HCO₃^-] maintain sperm quiescent during maturation in the epididymis. The vacuolar H⁺-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that PKA, acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO₃^-, induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer as compared with controls perfused without drug. Moreover, pre-perfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N⁶-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [³²P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNAi-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.