The oxacillinase gene was reported to confer limited resistance to carbapenem in Acinetobacter baumannii. In this study, we have demonstrated that an A. baumannii clinical isolate harboring a plasmid, pTVICU53, has 11,037 bp encoding 13 open reading frames. A bla(OXA-58) gene with an upstream insertion of truncated ISAba3 (DeltaISAba3) and IS1008 was found in this plasmid. DeltaISAba3and IS1008 provided two independent promoters for the transcription control of the bla(OXA-58) gene. The transformation of pTVICU53 or a shuttle vector bearing IS1008-DeltaISAba3-bla(OXA-58) to different A. baumannii recipients can increase their MICs of carbapenem 64- to 256-fold. The deletion of promoters provided by IS1008 resulted in dramatic decreases in bla(OXA-58) transcription and a 32- to 64-fold reduction in the carbapenem MIC. These findings highlight that A. baumannii might develop carbapenem resistance with a single transformation step, taking up a plasmid containing a genetic construct with a potentially high level of transcription of the bla(OXA-58) gene.