ExoU is a potent phospholipase A2 effector protein secreted by the type III secretion system of Pseudomonas aeruginosa. By cleaving plasma membrane phospholipids, it causes rapid lysis of eukaryotic cells. However, ExoU does not exhibit activity on its own but instead requires eukaryotic cell cofactors for activation. Ubiquitin and ubiquitinated proteins have been shown to activate ExoU, but previous work suggested that other cofactors are also involved. In this study, we demonstrate that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is another important coactivator of ExoU. PI(4,5)P2 works synergistically with ubiquitin to greatly enhance the phospholipase A2 activity of ExoU. Distinct residues of ExoU were critical for activation by PI(4,5)P2 and by ubiquitin, indicating that these factors activate ExoU by discrete mechanisms. In support of the biological relevance of PI(4,5)P2 coactivation, a yeast mutant with reduced PI(4,5)P2 levels was less susceptible to the cytotoxic activity of ExoU. Together, these findings further elaborate the molecular mechanism of ExoU.