Acute lung injury (ALI) is associated with an inflammation-mediated process, and the transcription factor, Krüppel-like factor 5 (KLF5), might play a crucial role in inflammatory lung disease. In this study, we evaluated KLF5, reactive oxygen species (ROS), and inflammatory responses in a lipopolysaccharide- (LPS-) induced ALI model to elucidate the role of KLF5 in ALI. Our data indicated that LPS upregulates proinflammatory cytokine expression in human bronchial epithelial cells in a dose-dependent manner. We observed upregulated KLF5 protein expression in human bronchial epithelial cells exposed to LPS, with peak expression 1 h after LPS treatment, and subsequent upregulation of p65 protein expression and p65 phosphorylation at Ser276. These results indicate that KLF5 mediates proinflammatory cytokine expression by upregulating nuclear factor-kappaB (NF-κB) phosphorylation at p65 in response to LPS. LPS treatment also increased ROS production and simultaneously upregulated KLF5 expression and NF-κB translocation. N-acetylcysteine significantly reduced ROS levels and KLF5 and NF-κB translocation in nuclear extracts. Therefore, N-acetylcysteine pretreatment before LPS exposure reduces ROS, downregulates KLF5 expression, and subsequently reduces inflammatory responses by scavenging ROS. Overall, our study results indicate that KLF5 mediates proinflammatory cytokine expression through upregulation of NF-κB phosphorylation at p65 in LPS-induced ALI.