BACKGROUND: Pseudomonas aeruginosa (PA) is a major pathogen that causes pneumonia and acute lung injury (ALI). Dysregulated NAT10 expression is associated with inflammatory and infectious diseases, but its role in PA-induced ALI remains unclear. METHODS: A mouse pneumonia model was established by intratracheal injection of PA, and lentivirus-mediated NAT10 interference and HMGB1 overexpression vectors were administered via the tail vein. Lung mechanics, protein content, total cell counts, neutrophil counts, inflammatory factor levels in bronchoalveolar lavage fluid (BALF), and lung bacterial load were assessed 24 h after PA injection. HE staining was performed to evaluate lung structural damage. Intracellular oxidative stress levels in mouse lung epithelial cells (TC-1 cells) were measured by detecting ROS and MDA levels. Mitochondrial function was analyzed by testing the mitochondrial membrane potential, cytoplasmic accumulation of cytochrome C, mtDNA copy number, and ATP production. An N4-acetylcytidine (ac4C)-RNA immunoprecipitation assay was conducted to assess the ac4C level of HMGB1 mRNA. RESULTS: NAT10 deficiency hindered PA infection-induced increases in immune cell infiltration, inflammatory factor levels, bacterial load, and ultimately lung structural and functional damage. However, upregulation of HMGB1 effectively antagonized the protective effects of NAT10 silencing in vivo. NAT10 knockdown suppressed PA-induced oxidative stress, mitochondrial dysfunction, and apoptosis in vitro. Whereas, HMGB1 overexpression reversed the inhibitory effects of NAT10 downregulation on PA-induced TC-1 cell injury. Mechanistically, as an acetyltransferase, NAT10 enhanced HMGB1 mRNA stability and protein expression by promoting HMGB1 mRNA ac4C modification. CONCLUSION: NAT10 facilitated mitochondrial dysfunction in lung epithelial cells and exacerbated PA-induced ALI by promoting the N4-acetylcytidine of HMGB1 mRNA.