Tigecycline is regarded as a last-resort treatment for multidrug-resistant Acinetobacter baumannii. However, tigecycline resistance in A. baumannii has increased worldwide. In this study, we investigated tigecycline heteroresistance in A. baumannii isolates from South Korea. Antibiotic susceptibility testing was performed on 323 nonduplicated A. baumannii isolates. Among 260 and 37 tigecycline-susceptible and -intermediate-resistant A. baumannii isolates, 146 (56.2%) and 22 (59.5%) isolates were identified as heteroresistant to tigecycline through a disk diffusion assay and population analysis profiling. For selected isolates, an time-kill assay was performed, and survival rates were measured after preincubation with diverse concentrations of tigecycline. Heteroresistant isolates showed regrowth after 12 h of 2× MIC of tigecycline treatment, and resistant subpopulations were selected by preexposure to tigecycline. Furthermore, genetic alterations in , and were assessed, and the relative mRNA expression levels of and were compared. The tigecycline resistance in subpopulations might be due to the insertion of IS in , leading to the overexpression of the AdeABC efflux pump. However, the tigecycline resistance of subpopulations was not stable during serial passages in antibiotic-free medium. The reversion of tigecycline susceptibility by antibiotic-free passages might occur by additional insertions of IS in and nucleotide alterations in in some mutants. Tigecycline heteroresistance is prevalent in A. baumannii isolates, which results in treatment failure. Tigecycline resistance is mainly due to the overexpression of the AdeABC efflux pump, which is associated with genetic mutations, but this resistance could be reversed into susceptibility by additional mutations in antibiotic-free environments. The evidence that antibiotic heteroresistance is responsible for treatment failure in clinical settings is increasing. Thus, detection and characterization of heteroresistance would be important for appropriate therapeutic guidance to treat bacterial infections. However, data on tigecycline heteroresistance in Gram-negative bacteria is currently limited, although tigecycline is regarded as a last-line antibiotic against infections caused by antibiotic-resistant pathogens. In this study, we investigated the tigecycline heteroresistance in Acinetobacter baumannii, which has been listed by the WHO as a priority for research and development of new antibiotics. We found very high prevalence of tigecycline-heteroresistant A. baumannii clinical isolates, which may result in treatment failure due to the selection of resistant subpopulations. We also identified the main resistance mechanism in tigecycline-resistant subpopulations, that is, upregulation of AdeABC efflux pumps due to IS insertion in