The phenomenon of colistin dependence in Acinetobacter baumannii has been described in a situation in which colistin is now considered as the last resort for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. In this study, we aimed to reveal a gene associated with colistin dependence in A. baumannii. The colistin-dependent A. baumannii H08-391D strain was isolated from a patient, and target gene-inactivation mutants were constructed. We investigated the effects of target gene on colistin dependence with quantitative real-time PCR and endotoxin assay. Also, we observed the change of cell morphology by electron microscopy. The expression of ACICU_02898, encoding a soluble lytic transglycosylase associated with cell-wall degradation and recycling, was increased by eight-to 42-fold in colistin-dependent mutants, and deletion of ACICU_02898 in a colistin-dependent strain led to colistin susceptibility (MIC = 8 mg/L). Endotoxin activity was significantly low in a colistin-dependent derivative ACICU_02898-inactivated mutant and a complemented mutant. In addition, the ACICU_02898-inactivated mutant showed a highly reduced growth rate. The colistin-dependent derivative and ACICU_02898-inactivated mutant showed clearly distinguished absorption profiles in the red/green fluorescence dot blot with regard to their membrane potential. Electron microscopy revealed that the deletion mutant cells were elongated compared to the colistin-susceptible wild-type strain and colistin-dependent strain. A colistin-dependent A. baumannii strain exhibited a deficiency in its outer membrane integrity and high expression of lytic transglycosylase was required for survival. This study reveals why the colistin-dependent mutant can tolerate high antibiotic concentrations.