Biotic stresses induced by herbivores result in diverse physiological changes in plants. In the interaction between the Lima bean (Phaseolus lunatus) and the herbivore Spodoptera littoralis, the earliest event induced by feeding on leaves is the depolarization of the plasma membrane potential (Vm), which is the results of both mechanical damage and insect oral secretions (OS). Although this herbivore-induced Vm depolarization depends on a calcium-dependent opening of potassium channels, the attacked leaf remains depolarized for an extended period, which cannot be explained by the sole action of potassium channels. Here we show that the plasma membrane H+-ATPase of P. lunatus leaves is strongly inhibited by S. littoralis OS. Inhibition of the H+-ATPase was also found in plasma membranes purified from leaf sections located distally from the application zone of OS, thus suggesting a long-distance transport of a signaling molecule(s). S. littoralis' OS did not influence the amount of the plasma membrane H+-ATPase, whereas the levels of membrane-bound 14-3-3 proteins were significantly decreased in membranes purified from treated leaves. Furthermore, OS strongly reduced the in vitro interaction between P. lunatus H+-ATPase and 14-3-3 proteins. The results of this work demonstrate that inhibition of the plasma membrane H+-ATPase is a key component of the S. littoralis OS mechanism leading to an enduring Vm depolarization in P. lunatus wounded leaves.