Abstract: OBJECTIVE To explore the mechanism of action of ROS/HIF-1α axis-mediated oxidative stress in the induction of cell barrier damage by multidrug-resistant Acinetobacter baumannii (MRAB). METHODS We developed a bronchial epithelial cell model infected with MRAB and set up a control and an MRAB infection groups. Based on Western blot and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), the levels of heme oxygenase-1 (HO-1), hypoxia-inducible factor-1α (HIF-1α), cell barrier function indicators occludin, zonula occludens-1 (ZO-1) and E-cadherin were compared between groups. The levels of reactive oxygen species (ROS) were detected by the DCFH-DA probe. Immunofluorescence technology was employed to detect the expression intensity and localization of key functional indicators. RESULTS MRAB exhibited a time- and concentration-dependent stimulatory effect on intracellular ROS release levels. Specifically, bacterial solution with a multiplicity of infection (MOI) of 10 could induce significant oxidative stress responses in infected cells after 6 hours (P<0.05). MRAB could reduce the activity of the antioxidant enzyme glutathione peroxidase (GPX) by activating HIF-1α and inhibiting HO-1 expression, thereby mediating intracellular ROS production (P<0.05). Inhibiting HIF-1α activity could alleviate the excessive activation of intracellular oxidative stress after infection and partially restore the cell barrier function (P<0.05). CONCLUSIONS MRAB activates oxidative stress levels and disrupts the barrier function of bronchial epithelial cells by activating the ROS/HIF-1α axis, thereby triggering pathogenic effects. This finding provides new insights for further clinical exploration of MRAB pathogenicity and the identification of therapeutic intervention targets.