Acute lung injury (ALI) is characterized by severely damaged alveoli and blood vessels, seriously affecting the health of patients and causing a high mortality rate. The pathogenesis of ALI is complex, with inflammatory reactions and oxidative stress (OS) mainly involved. S14G humanin (HNG) is derived from humanin (HN), which is claimed with promising anti-inflammatory functions. Herein, the protective influence of HNG on ALI will be explored in a mouse model. The ALI model was established in mice via intratracheal instillation of 3 mg/kg LPS, followed by an intraperitoneal injection of 3 and 6 mg/kg HNG, respectively. Thicker alveolar walls, aggravated neutrophil infiltration, and increased wet weight/dry weight (W/D) ratio were observed in ALI mice, accompanied by an aggravated apoptotic state, all of which were notably alleviated by HNG. Furthermore, increased number of total cells and neutrophils in bronchoalveolar lavage fluid (BALF), elevated secretion of inflammatory cytokines, enhanced reactive oxygen species (ROS) and Malondialdehyde (MDA) levels, and declined superoxide dismutase-2 (SOD2) levels were observed in ALI mice, which were markedly ameliorated by HNG. Moreover, the upregulated levels of NOD-like receptor family pyrin domain containing 3 (NLRP3), caspase-1, and caspases cleave gasdermin D N/caspases cleave gasdermin D FL (GSDMD N/GSDMD FL) in ALI mice were signally repressed by HNG. Lastly, the upregulation of Toll-like receptor 4 (TLR4) and p-p65/p65, and downregulation of IκB-α observed in ALI mice were sharply reversed by HNG. Collectively, HNG alleviated the ALI in mice by inhibiting the activation of nuclear factor kappa B (NF-κB) signaling.