Pseudomonas aeruginosa is an opportunistic pathogen that causes acute, invasive infections in immunocompromised individuals and chronic, persistent respiratory infections in individuals with cystic fibrosis (CF). The differential progression of acute or chronic infections involves the production of distinct sets of virulence factors. P. aeruginosa strains isolated from patients with acute respiratory infection are generally nonencapsulated and express a variety of invasive virulence factors, including flagella, the type III secretion system (T3SS), type IV pili (TFP), and multiple secreted toxins and degradative enzymes. Strains isolated from chronically infected CF patients, however, typically lack expression of invasive virulence factors and have a mucoid phenotype due to the production of an alginate capsule. The mucoid phenotype results from loss-of-function mutations in mucA, which encodes an anti-sigma factor that normally prevents alginate synthesis. Here, we report that the cyclic AMP/Vfr-dependent signaling (CVS) pathway is defective in mucA mutants and that the defect occurs at the level of vfr expression. The CVS pathway regulates the expression of multiple invasive virulence factors, including T3SS, exotoxin A, protease IV, and TFP. We further demonstrate that mucA-dependent CVS inhibition involves the alternative sigma factor AlgU (AlgT) and the response regulator AlgR but does not depend on alginate production. Our findings show that a single naturally occurring mutation leads to inverse regulation of virulence factors involved in acute and persistent infections. These results suggest that mucoid conversion and inhibition of invasive virulence determinants may both confer a selective advantage to mucA mutant strains of P. aeruginosa in the CF lung.