Treatment ofMycobacterium abscessuspulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem, meropenem).M. abscessusproduces a β-lactamase (BlaMab) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance inM. abscessusand poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collectedM. abscessussubsp.massilienseisolates from the index case of a CF center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC >512 µg/ml). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increasedblaMabmRNA level. Concurrent increase in transcription of precedingppiAgene indicated upregulation of the entire operon in the resistant strains. Deletion of the porinmspAcoincided with the first increase in MIC (from 8 to 32 µg/ml). A frameshift mutation inmsp2responsible for the rough colony morphology, and a SNP in ATP-dependent helicasehrpAco-occurred with the second increase in MIC (from 32 to 256 µg/ml). Increased BlaMabexpression and enzymatic activity may have been due to altered regulation of theppiA-blaMaboperon by the mutated HrpA alone, or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treatingM. abscessus, particularly imipenem resistant strains.