The monobactam scaffold is attractive for the development of new agents to treat infections caused by drug-resistant Gram-negative bacteria because it is stable to metallo-β-lactamases (MBLs). However, the clinically used monobactam aztreonam lacks stability to serine β-lactamases (SBLs) that are often coexpressed with MBLs. LYS228 is stable to MBLs and most SBLs. LYS228 bound purified Escherichia coli penicillin binding protein 3 (PBP3) similarly to aztreonam (derived acylation rate/equilibrium dissociation constant [ k 2/ K d ] of 367,504 s−1 M−1 and 409,229 s−1 M−1, respectively) according to stopped-flow fluorimetry. A gel-based assay showed that LYS228 bound mainly to E. coli PBP3, with weaker binding to PBP1a and PBP1b. Exposing E. coli cells to LYS228 caused filamentation consistent with impaired cell division. No single-step mutants were selected from 12 Enterobacteriaceae strains expressing different classes of β-lactamases at 8× the MIC of LYS228 (frequency, <2.5 × 10−9). At 4× the MIC, mutants were selected from 2 of 12 strains at frequencies of 1.8 × 10−7 and 4.2 × 10−9. LYS228 MICs were ≤2 μg/ml against all mutants. These frequencies compared favorably to those for meropenem and tigecycline. Mutations decreasing LYS228 susceptibility occurred in ramR and cpxA ( Klebsiella pneumoniae ) and baeS ( E. coli and K. pneumoniae ). Susceptibility of E. coli ATCC 25922 to LYS228 decreased 256-fold (MIC, 0.125 to 32 μg/ml) after 20 serial passages. Mutants accumulated mutations in ftsI (encoding the target, PBP3), baeR , acrD , envZ , sucB , and rfaI . These results support the continued development of LYS228, which is currently undergoing phase II clinical trials for complicated intraabdominal infection and complicated urinary tract infection (registered at ClinicalTrials.gov under identifiers NCT03377426 and NCT03354754).