Background: Glutathione-S-Transferase (GST) family of enzymes serve as a key antioxidant system. Clinical evidence suggests that several GST polymorphisms are associated with inflammatory lung diseases. The mechanisms by which these polymorphisms impact on cellular responses to stress remain unknown. To define the specific role of GST isoenzymes, we investigated the influence of GST inhibition on lung cell survival and inflammation, in in vitro and ex vivo models. Methodology: Pulmonary cells were exposed to GST inhibitors ethacrynic acid or caffeic acid alone, or in combination with oxidative stressors hydrogen peroxide, tert-butyl-hydroperoxide or hypoxia and reoxygenation. RNA interference was employed to attenuate specific GST isoform activity. Cell injury was assessed by MTT assay and flow cytometry. Oxidative stress was investigated by: (a) DHR123 and DCF fluorescence, (b) monitoring intracellular redox state by HPLC, (c) measurement of protein carbonyls by spectrophotometry and d) evaluation of the effects of specific and broad-spectrum antioxidants. Mitochondrial energetics was assessed by calculation of ATP/ADP ratios by HPLC, while global metabolic response was evaluated by NMR spectroscopy. An ex vivo mouse isolated perfused lung (IPL) model was used to explore the physiological role of GST. Principal findings: Two GST inhibitors and genetic attenuation of GSTπ significantly increased oxidative stress and induced cytotoxicity of lung epithelial cells and rendered them more susceptible to oxidative stress. While cellular energy state was not compromised, EA potentiated protein carbonylation and caused metabolic derangements. Furthermore, in the ex vivo model, EA significantly increased pulmonary permeability and intra-alveolar oedema, which may have been responsible for the increase in pulmonary elastance and resistance that were also observed. Conclusion: These observations highlight the importance of GST enzymes, specifically GSTπ, in the cellular and whole lung response to stress conditions and may help to understand the metabolic determinants of oxidative lung injury and adaptation.