Pseudomonas aeruginosa uses a complex type III secretion system to inject the toxins ExoS, ExoT, ExoU, and ExoY into the cytosol of target eukaryotic cells. This system is regulated by the exoenzyme S regulon and includes the transcriptional activator ExsA. Of the four toxins, ExoU is characterized as the major virulence factor responsible for alveolar epithelial injury in patients with P. aeruginosa pneumonia. Virulent strains of P. aeruginosa possess the exoU gene, whereas non-virulent strains lack this particular gene. The mechanism of virulence for the exoU+ genotype relies on the presence of a pathogenic gene cluster (PAPI-2) encoding exoU and its chaperone, spcU. The ExoU toxin has a patatin-like phospholipase domain in its N-terminal, exhibits phospholipase A2 activity, and requires a eukaryotic cell factor for activation. The C-terminal of ExoU has a ubiquitinylation mechanism of activation. This probably induces a structural change in enzymatic active sites required for phospholipase A2 activity. In P. aeruginosa clinical isolates, the exoU+ genotype correlates with a fluoroquinolone resistance phenotype. Additionally, poor clinical outcomes have been observed in patients with pneumonia caused by exoU+-fluoroquinolone-resistant isolates. Therefore, the potential exists to improve clinical outcomes in patients with P. aeruginosa pneumonia by identifying virulent and antimicrobial drug-resistant strains through exoU genotyping or ExoU protein phenotyping or both.