Updated project metadata. Hypoxia is a feature of the microenvironment during P. aeruginosa infection in several disease states. We confirm that the pathogenicity of P. aeruginosa derived from sites of acute infection is higher than those derived from sites of chronic infection. Hypoxia attenuated the pathogenicity of acute but not chronic strains implicating a role for hypoxia in controlling virulence. Mass spectrometric analysis revealed reduced expression of multiple virulence factors in hypoxia including exotoxin A, alkaline protease and proteins important in the synthesis of pyoverdine. Inhibition of pyoverdine production by iron supplementation mimicked the effects of hypoxia. Finally, strains of P. aeruginosa which lacks a functional pseudomonas prolyl-hydroxylase domain containing protein (PPHD) do not respond to hypoxia implicating a possible role for PPHD as an oxygen-sensing determinant of pathogenicity. Understanding how hypoxia influences bacterial virulence will identify new targets for anti–infective therapy against P. aeruginosa.