Abstract Background The emergence of multidrug-resistant Acinetobacter baumannii as a nosocomial pathogen is one of the major public health problems. Th e aim of this study was to evaluate the role of an efflux pump gene adeJ for the multidrug resistance of A. baumannii clinical isolates. Methods Two groups (MDRAB and SAB) of A. baum ann ii clinical isol a tes were studied. The SAB group consisted of strains that did not meet the criteria of MDRAB and were susceptible to more categories of antibiotics than MDRAB. Antimicrobial susceptibility results obtained by VITEKⅡ system were used in data analysis and bacterial group allocation. We performed real-time reverse transcription PCR to determine relative expression of adeJ . We compared relative expression of a deJ in comparison groups by considering two viewpoints: i) MDRAB and SAB groups and ii) susceptible and non-susceptible groups for each antibiotic used in this study. Results The mean value of relative expression of adeJ of MDRAB and SAB groups was 1.4 and 0.92, respectively, and showed significant diff e rence ( P =0.002). The mean values of relative expression of a deJ of susceptible and non-susceptible groups to the antibiotics cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem, tigecycline, piperacillin/ tazobactam, ticarcillin/clavulanic acid, trimethoprim/sulfamethoxazole, piperacillin, and gentamicin showed statistically significant differences. Conclusion The overexpression of adeIJK might contribute to the multidrug resistance in A. baumannii clinical isolates. Further, th e overexpression of adeIJK might be one of the factors contribut i ng to the resistance to numerous antibiotics.