Ferric uptake regulator (Fur) is a key regulatory factor of iron metabolism and virulence inPseudomonas aeruginosa. Many research groups have failed to construct thefur-deleted mutant ofP.aeruginosa, sofur has always been considered to be an essential gene inP.aeruginosa, and the knowledge of its biological function is limited. [Objective] This study aims to construct afur-deleted mutant ofP.aeruginosa and analyze its phenotypes. [Methods] WithP.aeruginosa PAO1 as the parental strain, thefur-deleted mutant was constructed by homologous recombination. After that, we studied the effects offur on the growth, siderophore biosynthesis, resistance to oxygen stress, flagella formation, biofilm formation, and virulence ofP.aeruginosa. In addition, we explored the cause of the growth defect phenotype of thefur-deleted mutant by genetic analysis. [Results] Thefur-deleted mutant ofP.aeruginosa was successfully constructed. The deletion offur greatly limited the growth ofP.aeruginosa and reduced the growth adaptability ofP.aeruginosa to the iron-limited environment, while it did not affect the growth adaptability ofP.aeruginosa to the iron-rich environment. This growth defect phenotype of Δfur was caused by the slow cell growth and proliferation, rather than by cell death. Interestingly, heterologousfur could completely complement the growth defect phenotype of Δfur, suggesting that the Fur ofP.aeruginosa was not functionally unique. Although there was a functional relationship between Fur and the toxin-antitoxin system PacTA, the growth defect phenotype ofP.aeruginosa Δfur was not associated with PacT toxin. In addition to affecting the growth phenotype ofP.aeruginosa, the deletion offur also madeP.aeruginosa lose the inhibitory effect on siderophore biosynthesis and the ability to form flagella and have increased sensitivity to H₂O₂ and reduced virulence toGalleria mellonella larvae. Moreover, the deletion offur increased the intracellular cyclic diguanylate (c-di-GMP) level ofP.aeruginosa to induce the expression ofpelF andpslA, thereby promoting the biofilm formation ofP.aeruginosa. [Conclusion] fur is a non-essential gene that can be deleted and plays a crucial role in the normal growth, siderophore biosynthesis, resistance to oxygen stress, flagellum formation, biofilm formation, and virulence ofP.aeruginosa, which lays a foundation for the development of vaccines and agents againstP.aeruginosa.