This study explores the protective effects and mechanistic pathways of imeglimin in models of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Imeglimin administration significantly enhanced survival rates, mitigated pulmonary edema, and attenuated histopathological lung damage in murine subjects. Furthermore, imeglimin decreased inflammatory cell infiltration, reduced protein leakage, lowered oxidative stress markers such as malondialdehyde, suppressed pro-inflammatory cytokines including IL-6 and CXCL2, and elevated the activity of the antioxidant enzyme superoxide dismutase within bronchoalveolar lavage fluid. In vitro studies using human pulmonary microvascular endothelial cells revealed that imeglimin preserved mitochondrial integrity and membrane potential, diminished reactive oxygen species production, and inhibited the opening of the mitochondrial permeability transition pore. Transcriptomic analyses identified TXNIP as a critical gene downregulated by imeglimin via enhanced methylation of its promoter region. This epigenetic modulation correlated with increased expression and activity of DNMT3B, and decreased activities of TET3. Molecular docking studies further substantiated the direct binding of imeglimin to DNMT3B and TET3, suggesting that imeglimin facilitates TXNIP promoter methylation by modulating interactions among DNMT3B, TET3, and the TXNIP promoter. Comparative analyses with seven other hypoglycemic agents demonstrated that imeglimin conferred the lowest mortality rate and hypoglycemia incidence in ALI mice, underscoring its superior therapeutic efficacy and safety. Collectively, these results indicate that imeglimin confers protection against LPS-induced ALI by promoting TXNIP promoter methylation, silencing TXNIP expression, enhancing mitochondrial quality control, and attenuating oxidative stress and inflammation, thereby improving survival outcomes and positioning imeglimin as a promising therapeutic candidate for ALI management.