Bacteria invest significant resources into the continuous creation and tailoring of their essential protective peptidoglycan (PG) cell wall. Several soluble PG biosynthesis products in the periplasm are transported to the cytosol for recycling, leading to enhanced bacterial fitness. GlcNAc-1,6-anhydroMurNAc and peptide variants are transported by the essential major facilitator superfamily importer AmpG in Gram-negative pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Accumulation of GlcNAc-1,6-anhydroMurNAc-pentapeptides also results from β-lactam antibiotic induced cell wall damage. In some species, these products upregulate the β-lactamase AmpC, which hydrolyzes β-lactams to allow for bacterial survival and drug-resistant infections. Here, we have used cryo-electron microscopy and chemical synthesis of substrates in an integrated structural, biochemical, and cellular analysis of AmpG. We show how AmpG accommodates the large GlcNAc-1,6-anhydroMurNAc peptides, including a unique hydrophobic vestibule to the substrate binding cavity, and characterize residues involved in binding that inform the mechanism of proton-mediated transport.Antimicrobial resistance (AMR) is an escalating global health crisis, with the evolution of resistance outpacing drug development 1 . βlactams represent over 65% of all antibiotics prescribed globally and play a critical role in infectious disease management, but their efficacy is threatened by multiple mechanisms of resistance 2 . While there is an urgent need for new strategies to address AMR, understanding the current mechanisms of β-lactam resistance enables creation of complementary therapeutics administered as cocktail treatments, prolonging the effectiveness of many existing antibiotics approved against various bacterial species and pathologies. β-lactams covalently bind and inhibit the transpeptidation domain of penicillin-binding proteins (PBPs). This interrupts the essential peptide cross-linking of