In Pseudomonas aeruginosa, tryptophan can be converted into anthranilate via the KynABU pathway, and anthranilate as a substrate is further converted into alkyl quinolones (AQs), including 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ), under the action of pqsABCDE, a synthetic gene cluster of AQs. At the same time, anthranilate can be degraded into the tricarboxylic acid cycle under the catalysis of the anthranilate dioxygenase complex AntABC, while the biological effect of AntABC on P. aeruginosa remains unclear. [Objective] To construct and characterize the phenotype of the antABC-deleted mutant of P. aeruginosa. [Methods] With P. aeruginosa PAO1 as the starting strain, we constructed the antABC-deleted mutant by homologous recombination to study the effects of the operon on tryptophan degradation, biofilm formation, pyocyanin synthesis, motility, and virulence of P. aeruginosa. [Results] The deletion of antABC or kynU completely inhibited the growth of P. aeruginosa with tryptophan as the sole carbon source, while ΔpqsA did not present this phenotype, indicating that antABC was essential for the degradation of tryptophan by P. aeruginosa, and KynABU-AntABC pathway was the only way for the degradation of tryptophan by the bacterium under the culture conditions in this study. In addition to affecting tryptophan degradation in P. aeruginosa, the deletion of antABC promoted the biofilm formation of P. aeruginosa by inducing the expression of the extracellular polysaccharide synthesis operon pel, and it promoted the synthesis of pyocyanin by inducing the expression of the pyocyanin synthesis operons phz1 and phz2. In addition, the deletion of antABC enhanced the swarming motility and twitching motility of P. aeruginosa. Interestingly, further deletion of pqsA completely reversed the physiological phenotypes of ΔantABC. Therefore, the regulation of antABC on these physiological phenotypes depended on AQs. The deletion of antABC increased the HHQ accumulation while inhibiting the synthesis of PQS in P. aeruginosa. These results indicated that the regulation of these physiological phenotypes by antABC mainly depended on HHQ. In addition, the deletion of antABC enhanced the virulence of P. aeruginosa to Chinese cabbage and Galleria mellonella larvae, while further deletion of pqsA only partially reversed this virulence phenotype. Moreover, the deletion of antABC caused increased accumulation of anthranilate in P. aeruginosa. Therefore, the enhancement of antABC deletion on the virulence of P. aeruginosa was mediated by HHQ and anthranilate together. Finally, bioinformatics analysis revealed that the missense mutations of antABC operon occurred in more than 90% of clinical isolates of P. aeruginosa. Therefore, antABC was expected to be used as a biomarker to determine whether clinical isolates of P. aeruginosa were highly virulent. [Conclusion] AntABC plays an important role in the tryptophan degradation, biofilm formation, pyocyanin synthesis, motility, and virulence of P. aeruginosa. This finding lays a foundation for the clinical diagnosis and antimicrobial development of P. aeruginosa infection.