Nepenthes is a unique genus of carnivorous plants that can capture insects in trapping organs called pitchers and digest them in pitcher fluid. The pitcher fluid includes digestive enzymes and is strongly acidic. We found that the fluid pH decreased when prey accumulates in the pitcher fluid of Nepenthes alata. The pH decrease may be important for prey digestion and the absorption of prey-derived nutrients. To identify the proton pump involved in the acidification of pitcher fluid, plant proton-pump homologs were cloned and their expressions were examined. In the lower part of pitchers with natural prey, expression of one putative plasma-membrane (PM) H+-ATPase gene, NaPHA3, was considerably higher than that of the putative vacuolar H+-ATPase (subunit A) gene, NaVHA1, or the putative vacuolar H+-pyrophosphatase gene, NaV-HP1. Expression of one PM H+-ATPase gene, Na-PHA1, was detected in the head cells of digestive glands in the lower part of pitchers, where proton extrusion may occur. Involvement of the PM H+-ATPase in the acidification of pitcher fluid was also supported by experiments with proton-pump modulators; vanadate inhibited proton extrusion from the inner surface of pitchers, whereas bafilomycin A1 did not, and fusicoccin induced proton extrusion. These results strongly suggest that the PM H+-ATPase is responsible for acidification of the pitcher fluid of Nepenthes.