Sepsis-induced acute lung injury (ALI) is a serious complication that substantially increases mortality in sepsis. Glycerol-3-phosphate dehydrogenase 2 (GPD2), a mitochondrial enzyme, is involved in oxidative stress, inflammation, and ferroptosis. However, its specific role in sepsis-induced ALI remains unclear. This study explored the role of GPD2 in sepsis-induced ALI. The study employed a cecal ligation and puncture (CLP) mouse model of sepsis, with GPD2 expression knocked down using adeno-associated virus vectors. Selenomethionine (SeMet), a GPX4 activator, was administered 24 h prior to CLP surgery. Increased GPD2 expression was observed in the lungs of septic mice. GPD2 knockdown mice subjected to CLP had a reduced survival rate and exhibited exacerbated lung injury, characterized by increased vascular permeability, neutrophil infiltration, inflammation, and oxidative stress. Moreover, GPD2 knockdown significantly promoted ferroptosis in the lungs of septic mice. Notably, SeMet not only inhibited ferroptosis but also partially reversed the lung injury exacerbated by GPD2 knockdown in septic mice. Our results suggest that GPD2 may protect against pulmonary vascular permeability, neutrophil infiltration, oxidative stress, and inflammatory responses in septic mouse lung tissues by suppressing ferroptosis through the activation of the GPX4 pathway. These findings suggest that GPD2 could be a potential therapeutic target for sepsis-induced ALI.