ABSTRACTExopolysaccharide Psl is a critical biofilm matrix component inPseudomonas aeruginosa, which forms a fiber-like matrix to enmesh bacterial communities. Iron is important forP. aeruginosabiofilm development, yet it is not clearly understood how iron contributes to biofilm development. Here, we showed that iron promoted biofilm formation via elevating Psl production inP. aeruginosa. The high level of iron stimulated the synthesis of Psl by reducing rhamnolipid biosynthesis and inhibiting the expression of AmrZ, a repressor ofpslgenes. Iron-stimulated Psl biosynthesis and biofilm formation held true in mucoidP. aeruginosastrains. Subsequent experiments indicated that iron bound with Pslin vitroand in biofilms, which suggested that Psl fibers functioned as an iron storage channel inP. aeruginosabiofilms. Moreover, among three matrix exopolysaccharides ofP. aeruginosa, Psl is the only exopolysaccharide that can bind with both ferrous and ferric ion, yet with higher affinity for ferrous iron. Our data suggest a survival strategy ofP. aeruginosathat uses exopolysaccharide to sequester and store iron to stimulate Psl-dependent biofilm formation.IMPORTANCEPseudomonas aeruginosais an environmental microorganism which is also an opportunistic pathogen that can cause severe infections in immunocompromised individuals. It is the predominant airway pathogen causing morbidity and mortality in individuals affected by the genetic disease cystic fibrosis (CF). Increased airway iron and biofilm formation have been proposed to be the potential factors involved in the persistence ofP. aeruginosain CF patients. Here, we showed that a high level of iron enhanced the production of the key biofilm matrix exopolysaccharide Psl to stimulate Psl-dependent biofilm formation. Our results not only make the link between biofilm formation and iron concentration in CF, but also could guide the administration or use of iron chelators to interfere with biofilm formation inP. aeruginosain CF patients. Furthermore, our data also imply a survival strategy ofP. aeruginosaunder high-iron environmental conditions.