Sivelestat sodium (SIV), a neutrophil elastase inhibitor, is mainly used for the clinical treatment of acute respiratory distress syndrome (ARDS) or acute lung injury (ALI). However, studies investigating the effects of SIV treatment of ALI are limited. Therefore, this study investigated the potential molecular mechanism of the protective effects of SIV against ALI. Human pulmonary microvascular endothelial cells (HPMECs) were stimulated with tumor necrosis factor α (TNF-α), and male Sprague-Dawley rats were intratracheally injected with Klebsiella pneumoniae (KP) and treated with SIV, ML385, and anisomycin (ANI) to mimic the pathogenetic process of ALI in vitro and in vivo, respectively. The levels of inflammatory cytokines and indicators of oxidative stress were assessed in vitro and in vivo. The wet/dry (W/D) ratio of lung tissues, histopathological changes, inflammatory cells levels in bronchoalveolar lavage fluid (BALF), and survival rates of rats were analyzed. The JNK/NF-κB (p65) and Nrf2/HO-1 levels in the HPMECs and lung tissues were analyzed by western blot and immunofluorescence analyses. Administration of SIV reduced the inflammatory factors levels, intracellular reactive oxygen species (ROS) production, and malondialdehyde (MDA) levels and increased the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in lung tissues. Meanwhile, SIV alleviated pathological injuries, decreased the W/D ratio, and inflammatory cell infiltration in lung tissue. In addition, SIV also inhibited the activation of JNK/NF-κB signaling pathway, promoted nuclear translocation of Nrf2, and upregulated the expression of heme oxygenase 1 (HO-1). However, ANI or ML385 significantly reversed these changes. SIV effectively attenuated the inflammatory response and oxidative stress. Its potential molecular mechanism was related to the JNK/NF-κB activation and Nrf2/HO-1 signaling pathway inhibition. This further deepened the understanding of the protective effects of SIV against ALI.