Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals. Small regulatory RNAs (sRNAs) regulate multiple bacterial adaptations to environmental changes, especially virulence. Our previous study showed that sRNA PrrH negatively regulates the expression of a number of virulence factors, such as pyocyanin, rhamnolipid, biofilm, and elastase in the P. aeruginosa strain PAO1. However, previous studies have shown that the prrH-deficient mutant attenuates virulence in an acute murine lung infection model. All Delta prrH-infected mice survived the entire 28-day course of the experiment, whereas all mice inoculated with the wild-type or the complemented mutant succumbed to lung infection within 4 days of injection, but the specific mechanism is unclear. Herein, we explored how PrrH mediates severe lung injury by regulating the expression of virulence factors. In vivo mouse and in vitro cellular assays demonstrated that PrrH enhanced the pathogenicity of PAO1, causing severe lung injury. Mechanistically, PrrH binds to the coding sequence region of the mRNA of exsA, which encodes the type III secretion system master regulatory protein. We further demonstrated that PrrH mediates a severe inflammatory response and exacerbates the apoptosis of A549 cells. Overall, our results revealed that PrrH positively regulates ExsA, enhances the pathogenicity of P. aeruginosa, and causes severe lung injury. IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium and the leading cause of nosocomial pneumonia. The pathogenicity of P. aeruginosa is due to the secretion of many virulence factors. Small regulatory RNAs (sRNAs) regulate various bacterial adaptations, especially virulence. Therefore, understanding the mechanism by which sRNAs regulate virulence is necessary for understanding the pathogenicity of P. aeruginosa and the treatment of the related disease. In this study, we demonstrated that PrrH enhances the pathogenicity of P. aeruginosa by binding to the coding sequence regions of the ExsA, the master regulatory protein of type III secretion system, causing severe lung injury and exacerbating the inflammatory response and apoptosis. These findings revealed that PrrH is a crucial molecule that positively regulates ExsA. Type III-positive strains are often associated with a high mortality rate in P. aeruginosa infections in clinical practice. Therefore, this discovery may provide a new target for treating P. aeruginosa infections, especially type III-positive strains.