Many proteobacteria, such as Escherichia coli, contain two main types of quinones, benzoquinones represented by ubiquinone (UQ) and naphthoquinones such as menaquinone (MK) and dimethyl-menaquinone (DMK). MK and DMK function predominantly in anaerobic respiratory chains, whereas UQ is the major electron carrier in the reduction of dioxygen. However, this division of labor is probably not very strict. Indeed, a pathway that produces UQ under anaerobic conditions in an UbiU-, UbiV-, and UbiT-dependent manner has been recently discovered in E. coli. However, its physiological relevance is not yet understood because MK and DMK are also present in E. coli. Here, we established that UQ9 is the major quinone of Pseudomonas aeruginosa and is required for growth under anaerobic respiration (i.e. denitrification). We demonstrate that the ORFs PA3911, PA3912, and PA3913, which are homologs of the E. coli ubiT, ubiV and ubiU genes, respectively, are essential for UQ9 biosynthesis and thus for denitrification in P. aeruginosa. These three genes hereafter are called ubiTPa, ubiVPa, and ubiUPa. We show that UbiVPa accommodates an iron-sulfur [4Fe-4S] cluster. Moreover, we report that UbiUPa and UbiTPa can bind UQ and that the isoprenoid tail of UQ is the structural determinant required for recognition by these two Ubi proteins. Since the denitrification metabolism of P. aeruginosa is believed to be important for pathogenicity of this bacterium in individuals with cystic fibrosis, our results highlight that the O2-independent UQ biosynthetic pathway may represent a possible target for antibiotics development to manage P. aeruginosa infections.