We previously demonstrated that diperoxovanadate (DPV), a synthetic peroxovanadium compound and cell-permeable oxidant that acts as a protein tyrosine phosphatase inhibitor and insulinomimetic, increased phospholipase D (PLD) activation in endothelial cells (ECs). In this report, the regulation of DPV-induced PLD activation by mitogen-activated protein kinases (MAPKs) was investigated. DPV activated extracellular signal-regulated kinase, c-Jun NH2-terminal kinase (JNK), and p38 MAPK in a dose- and time-dependent fashion. Treatment of ECs with p38 MAPK inhibitors SB-203580 and SB-202190 or transient transfection with a p38 dominant negative mutant mitigated the PLD activation by DPV but not by phorbol ester. SB-202190 blocked DPV-mediated p38 MAPK activity as determined by activated transcription factor-2 phosphorylation. Immunoprecipitation of PLD from EC lysates with PLD1 and PLD2 antibodies revealed both PLD isoforms associated with p38 MAPK. Similarly, PLD1 and PLD2 were detected in p38 immunoprecipitates from control and DPV-challenged ECs. Binding assays demonstrated interaction of glutathione S-transferase-p38 fusion protein with PLD1 and PLD2. Both PLD1 and PLD2 were phosphorylated by p38 MAPK in vitro, and DPV increased phosphorylation of PLD1 and PLD2 in vivo. However, phosphorylation of PLD by p38 failed to affect PLD activity in vitro. These results provide evidence for p38 MAPK-mediated regulation of PLD in ECs.