Objective To explore the resistance mechanism of tigecycline insensitivity of multidrug-resistant Acinetobacter baumannii, and provide reference for clinical rational antimicrobial use as well as prevention and control of healthcare-associated infection. Methods 22 strains of tigecycline insensitive multidrug-resistant Acinetobacter baumannii (TIS-MDR-AB) and 22 strains of tigecycline sensitive multidrug-resistant Acinetobacter baumannii (TS-MDR-AB) isolated clinically from the First Affiliated Hospital of Guangzhou Medical University from April 2022 to May 2023 were collected. Efflux pump phenotype inhibition assay was performed using efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The main efflux pump genes (adeB, adeG, adeJ), as well as tigecycline-resistant gene tet (X), were screened by polymerase chain reaction (PCR) technique, and their mRNA expression levels were detected by real-time fluorescence quantitative PCR. Mutations in the efflux pump regulatory gene adeRS were analyzed by Sanger sequencing analysis. Results The detection rates of efflux pump genes adeB, adeG and adeJ were all above 95% in two MDR-AB groups, and tet (X) gene was not detected. Efflux pump inhibitor assay showed that the minimum inhibitory concentration (MIC) of TIS-MDR-AB strains decreased after adding CCCP, 3 strains showed positive efflux pump phenotype. The mRNA expression level of MDR-AB adeB in the TIS-MDR-AB group was higher than that in the TS-MDR-AB group (P<0.01), while the expression of adeG and adeJ genes was no statistically different. Multiple mutations were found in the adeR and adeS genes, the adeS of 2 strains was inserted ISAba1, and 3 strains were inserted ISAba13. Conclusion The overexpression of adeABC in the efflux pump system may play an important role in the mechanism of reduced sensitivity of MDR-AB to tigecycline, and its overexpression may be related to the insertion sequence or mutation in the efflux pump regulatory gene adeRS.