ABSTRACTUnderstanding the interplay between antibiotic resistance and bacterial fitness and virulence is essential to guide individual treatments and improve global antibiotic policies. A paradigmatic example of a resistance mechanism is the intrinsic inducible chromosomal β-lactamase AmpC from multiple Gram-negative bacteria, includingPseudomonas aeruginosa, a major nosocomial pathogen. The regulation ofampCexpression is intimately linked to peptidoglycan recycling, and AmpC-mediated β-lactam resistance is frequently mediated by inactivating mutations inampD, encoding anN-acetyl-anhydromuramyl-l-alanine amidase, affecting the levels ofampC-activating muropeptides. Here we dissect the impact of the multiple pathways causing AmpC hyperproduction onP. aeruginosafitness and virulence. Through a detailed analysis, we demonstrate that the lack of all threeP. aeruginosaAmpD amidases causes a dramatic effect in fitness and pathogenicity, severely compromising growth rates, motility, and cytotoxicity; the latter effect is likely achieved by repressing key virulence factors, such as protease LasA, phospholipase C, or type III secretion system components. We also show thatampCoverexpression is required but not sufficient to confer the growth-motility-cytotoxicity impaired phenotype and that alternative pathways leading to similar levels ofampChyperexpression and resistance, such as those involving PBP4, had no fitness-virulence cost. Further analysis indicated that fitness-virulence impairment is caused by overexpressingampCin the absence of cell wall recycling, as reproduced by expressingampCfrom a plasmid in an AmpG (muropeptide permease)-deficient background. Thus, our findings represent a major step in the understanding of β-lactam resistance biology and its interplay with fitness and pathogenesis.IMPORTANCEUnderstanding the impact of antibiotic resistance mechanisms on bacterial pathogenesis is critical to curb the spread of antibiotic resistance. A particularly noteworthy antibiotic resistance mechanism is the β-lactamase AmpC, produced byPseudomonas aeruginosa, a major pathogen causing hospital-acquired infections. The regulation of AmpC is linked to the cell wall recycling pathways, and frequently, resistance to β-lactams is caused by mutation of several of the components of the cell wall recycling pathways such as AmpD. Here we dissect the impact of the pathways for AmpC hyperproduction on virulence, showing that the lack of all threeP. aeruginosaAmpD amidases causes a major effect in fitness and pathogenicity, compromising growth, motility, and cytotoxicity. Further analysis indicated that fitness-virulence impairment is specifically caused by the hyperproduction of AmpC in the absence of cell wall recycling. Our work provides valuable information for delineating future strategies for combatingP. aeruginosainfections by simultaneously targeting virulence and antibiotic resistance.