Lipopolysaccharide (LPS)-induced lung injury (ALI) is characterized by severe lung inflammation with high mortality. Pulmonary inflammation and apoptosis of alveolar epithelial cells cause the disruption of alveolar epithelial cell integrity, resulting in the failure of gas exchange, which is correlated with poor prognosis. However, the underlying regulatory mechanism remains unclear. In this study, we found that the Krüppel-like transcription factor KLF14 can suppress the apoptosis of alveolar epithelial cells in a LPS-mediated murine acute lung injury model by western blot and TUNEL assays. First, we found that the expression of KLF14 in alveolar epithelial cells of a murine ALI model was upregulated, and KLF14 deletion exacerbated lung injury, increased mortality, and promoted the apoptosis of alveolar epithelial cells. Furthermore, we identified that KLF14 protects lung tissue from LPS-mediated damage and suppresses the apoptosis of alveolar epithelial cells by inhibiting the NF-κB pathway. Our study provides further research insight and helps to explore the protective mechanism of KLF14 in inflammation and cell death. Our results suggested that the transcription factor KLF14 may act as a new target for the prevention and treatment of acute lung injury.