Tandem duplication of carbapenemase genes amplifies the gene copy number and enhances carbapenem resistance. These amplifications are often heterogenous, transient, locate in plasmids, which often also contribute to heteroresistance. The duplication is especially important for low-hydrolysis activity enzymes, which is often overlooked or even under controversy. Here we reported an intrinsic oxacillinase duplication mediated by two neighbor ISAba1inserted in the same orientation. The duplication is relatively stable, located in chromosome, and the duplication times is up to twenty-five, much larger than previous reports. We provided genomic, transcriptomic, and proteomic evidence that the duplication resulted oxacillinase overproduction and thus contribute to carbapenem resistance. No other possible carbapenem resistance related changes (point mutations of oxaAb, disturbed expression of porin protein and efflux pump) were found during the duplication process. Furthermore, introducing oxaAb flanked by two ISAba1 to A. baumannii via plasmids, mimicked the in vivo duplication process under carbapenem stress. Taken together, ISAba1 mediated duplication of low activity intrinsic antibiotic hydrolysis enzymes could lead to antibiotic resistance for advanced-generation antibiotics.