Phospholipase Dα (PLDα) is involved in plant response to salt stress, but the mechanisms remain unclear. We investigated rice PLDα (OsPLDα) localization and its effect on tonoplast (TP) and plasma membrane (PM) H(+)-ATPase activity and transcription in response to NaCl. When rice suspension-cultured cells were treated with 100 mM NaCl, PLDα activity in cell extracts showed a transient activation with a threefold increase at 1 h. The amount of OsPLDα protein decreased slightly in the cytosolic fractions, whereas it increased significantly in the TP after NaCl treatment. OsPLDα1 knockdown cells were developed using RNA interference (RNAi) methods. The increase in TP and PM H(+)-ATPase activity induced by NaCl was significantly inhibited in OsPLDα1-RNAi cells. Knockdown of OsPLDα1 prevented the NaCl-induced increase in the transcript level of OsVHA-A (encodes TP H(+)-ATPase) and OSA2 (encodes PM H(+)-ATPase), as well as OsNHX1 (encodes TP Na(+) /H(+) antiporter). The cells died more in OsPLDα1-RNAi mutant than in wild type when they were treated with NaCl. These results suggest that OsPLDα is involved in salt tolerance in rice through the mediation of H(+)-ATPase activity and transcription.